Lymphocyte chemoattractant factor and uses thereof

ABSTRACT

Disclosed is a substantially pure antibody which specifically binds a LCF polypeptide and methods of using such antibodies.

This work was supported in part by a grant from the Federal Governmentand the Government therefore has certain rights in the invention.

BACKGROUND OF THE INVENTION

This is a divisional of application Ser. No. 08/480,156, filed on Jun.7, 1995; which is a continuation-in-part of U.S. Ser. No. 08/354,961,filed Dec. 13, 1994; which is a continuation of U.S. Serial No.08/068,949, filed May 21, 1993 which is now abandoned.

This invention relates to recombinant LCF, DNA, and uses thereof.

CD4, a cell-cell adhesion protein, is expressed on a subset of Tlymphocytes (Krensky et al., Proc. Natl. Acad. Sci. USA 79:2365-2369,1982; Biddison et al., J. Exp. Med. 156:1065-1076, 1982; and Wilde etal., J. Immunol. 131:152-157, 1983), mononuclear cells (Stewart et al.,J. Immunol. 136:3773-3778, 1986), and eosinophils (Rand et al., J. Exp.Med. 173:1521-1528, 1991). In lymphocytes, CD4 contributes to antigenreceptor signaling (Collins et al., J. Immunol. 148:2159-2162, 1992;Anderson et al., J. Immunol. 139-678-682, 1987; Eichmann et al., J.Immunol. 17:643-650, 1987; Walker et al., Eur. J. Immunol. 17:873-8801987; and Sleckman et al., Nature 328:351-353, 1987) by directinteraction with MHC Class II molecules (Doyle et al., Nature330:256-259, 1987). In addition, a natural soluble lymphokine,lymphocyte chemoattractant factor (LCF), requires cell surfaceexpression of CD4 to induce chemotactic activity in monocytes(Cruikshank et al., J. Exp. Med. 173:1521-1528, 1991) and T lymphocytes(Cruikshank et al., J. Immunol. 138:3817-3823, 1987; Cruikshank et al.,J. Immunol. 146:2928-2934, 1991). In concert with its chemoattractantactivity LCF acts as a competence growth factor for human T lymphocytes(Cruikshank et al., J. Immunol. 138:3817-3823, 1987).

LCF is a cationic, 56-kD glycoprotein representing the tetrameric formof four 14-kD monomeric chains. LCF is produced by T lymphocytes and isspecifically chemoattractant for CD4+ T-cells, monocytes and eosinophils(see, e.g., Berman et al. Cell Immunol. 95:105-112, 1985; Rand et al.,JEM 173:1521-1528, 1991). Secretion of LCF by CD8+ T cells occurs(Cruikshank et al., J. Immunol. 138:3817, 1987;) after stimulation bymitogen, antigen, histamine or serotonin. The latter two are ofparticular interest because degranulated mast cells and basophils arepresent in tissue sites of delayed-type hypersensitivity reactions (see,e.g., Askenase Prog. Allergy 23:199-320, 1977). Induction of LCF by amast cell or a basophil product provides a link between the earlymediator phase of the immune response and the development of the laterT-lymphocyte-predominant inflammatory reaction.

SUMMARY OF THE INVENTION

In general, the invention features recombinant lymphocytechemoattractant factor (LCF) polypeptide, e.g., LCF produced in aprokaryotic or baculovirus expression system. Preferably, thepolypeptide includes an amino acid sequence substantially identical tothe amino acid sequence shown in FIG. 2 (SEQ ID NO: 1). By "lymphocytechemoattractant factor polypeptide" is meant all or part of a proteinwhich specifically binds CD4 and signals the appropriate LCF-mediatedcascade of biological events, e.g., a polypeptide capable of promotingor stimulating the migration of unactivated or activated CD4⁺lymphocytes, eosinophils, monocytes, and the like. By "polypeptide" ismeant any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation). By a"substantially identical" amino acid sequence is meant an amino acidsequence which differs only by conservative amino acid substitutions,for example, substitution of one amino acid for another of the sameclass (e.g., valine for glycine, arginine for lysine and the like) or byone or more non-conservative amino acid substitutions, deletions, orinsertions located at positions of the amino acid sequence which do notdestroy the biological activity of the polypeptide. Such equivalentpolypeptides can be isolated by extraction from tissues or cells of anyanimal which naturally produce such a polypeptide or which can beinduced to do so, using the methods described below, or theirequivalent; or can be isolated by chemical synthesis; or can be isolatedby standard techniques of recombinant DNA technology, e.g., by isolationof cDNA or genomic DNA encoding such a polypeptide.

In another aspect, the invention features a fragment or analog of LCFwhich exhibits LCF agonist or antagonist activity. The invention thusincludes any biologically active fragment or analog of LCF polypeptide.By "biologically active" is meant possessing any activity which ischaracteristic of the 130-amino acid LCF polypeptide shown in FIG. 2(SEQ ID NO: 1). Because LCF polypeptide exhibits a range ofphysiological properties and because such properties may be attributableto different portions of the LCF polypeptide molecule, a useful LCFpolypeptide fragment or LCF polypeptide analog is one which exhibits abiological activity in any biological assay for LCF polypeptideactivity, for example, those assays described herein. Most preferably itpossesses 10%, preferably 40%, or at least 90% of the activity of LCFpolypeptide (shown in FIG. 2; SEQ ID NO: 1), in any LCF polypeptideassay.

Preferred analogs include LCF polypeptide (or biologically activefragments thereof) whose sequences differ from the wild-type sequenceonly by conservative amino acid substitutions, for example, substitutionof one amino acid for another with similar characteristics (e.g., valinefor glycine, arginine for lysine, and the like) or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not abolish the polypeptide's biological activity. Other usefulmodifications include those which increase peptide stability; suchanalogs may contain, for example, one or more non-peptide bonds (whichreplace the peptide bonds) or D-amino acids in the peptide sequence.

Analogs can differ from naturally occurring LCF polypeptide in aminoacid sequence or can be modified in ways that do not involve sequence,or both. Analogs of the invention will generally exhibit at least 70%,more preferably 80%, more preferably 90%, and most preferably 95% oreven 99%, homology with a segment of 20 amino acid residues, preferablymore than 40 amino acid residues, or more preferably the entire sequenceof a naturally occurring LCF polypeptide sequence.

Alterations in primary sequence include genetic variants, both naturaland induced. Also included are analogs that include residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring or synthetic amino acids, e.g., β or γ amino acids.Alternatively, increased stability may be conferred by cyclizing thepeptide molecule. Modifications include in vivo or in vitro chemicalderivatization of polypeptides, e.g., acetylation, methylation,phosphorylation, phremylation, isupremylation, myristilation,carboxylation, or glycosylation; glycosylation can be modified, e.g., bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps, e.g., byexposing the polypeptide to glycosylation affecting enzymes derived fromcells that normally provide such processing, e.g., mammalianglycosylation enzymes; phosphorylation can be modified by exposing thepolypeptide to phosphorylation-altering enzymes, e.g., kinases orphosphatases, etc. By "substantially pure" is meant that the LCFpolypeptide provided by the invention is at least 60%, by weight, freefrom the proteins and naturally-occurring organic molecules with whichit is naturally associated. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight, LCF polypeptide. A substantially pure LCF polypeptide may beobtained, for example, by extraction from a natural source (e.g., ahuman peripheral blood mononuclear cell) using the methods outlinedbelow; or can be isolated by expression of a recombinant nucleic acidencoding a LCF polypeptide using the standard techniques of recombinantDNA technology, e.g., by isolation of cDNA or genomic DNA encoding suchan LCF polypeptide, or by chemically synthesizing the protein, fragmentor analog thereof. Purity can be measured by any appropriate method,e.g., column chromatography, polyacrylamide gel electrophoresis, orhigh-performance liquid chromatography (HPLC) analysis.

In another aspect, the invention features substantially pure DNAencoding a LCF polypeptide (or polypeptide fragment or analog thereof)as described above. Preferably, the DNA comprises a nucleotide sequencesubstantially identical to the nucleotide sequence shown in FIG. 2 (SEQID NO: 2). Moreover, such a DNA is cDNA and encodes a mammalian LCFpolypeptide, e.g., a human. The invention also features a vector whichincludes such substantially pure DNA and which is capable of directingexpression of the protein encoded by the DNA in a vector-containingcell. The invention features a cell which contains the substantiallypure DNA. The cell may be either prokaryotic, e.g., E. coli oreukaryotic, e.g., a mammalian cell or the cell of an arthropod, e.g., agrasshopper.

By "substantially pure DNA" is meant DNA that is free of the geneswhich, in the naturally-occurring genome of the organism from which theDNA of the invention is derived, flank the gene. The term thereforeincludes, for example, a recombinant DNA which is incorporated into avector; into an autonomously replicating plasmid or virus; or into thegenomic DNA of a prokaryote or eukaryote; or which exists as a separatemolecule (e.g., a cDNA or a genomic or cDNA fragment produced bypolymerase chain reaction -(PCR) methodologies or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In another aspect, the invention features a method of producing arecombinant LCF polypeptide (or a fragment or analog thereof). Themethod involves (a) providing a cell (e.g., E. coli or S. frugideratransformed with DNA encoding a LCF polypeptide or a fragment or analogthereof positioned for expression in the cell; (b) culturing thetransformed cell under conditions for expressing the DNA; and (c)isolating the recombinant LCF polypeptide. By "transformed cell" ismeant a cell into which (or into an ancestor of which) has beenintroduced, by means of recombinant techniques, a DNA molecule encoding(as used herein) an LCF polypeptide. Such a DNA molecule is "positionedfor expression" meaning that the DNA molecule is positioned adjacent toa DNA sequence which directs transcription and translation of thesequence (i.e., facilitates the production of, e.g. LCF, or fragment oranalog thereof).

In still another aspect, the invention features a substantially pureantibody which binds preferentially to a LCF (or a fragment or analogthereof). By "substantially pure antibody" is meant antibody which is atleast 60%, by weight, free from the proteins and naturally-occurringorganic molecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and morepreferably at least 99%, by weight, antibody, e.g., LCF antibody. Asubstantially pure LCF antibody may be obtained, for example, byaffinity chromatography using recombinantly-produced LCF polypeptide andstandard techniques. Furthermore, the purified antibody is sufficientlyfree of other proteins, carbohydrates, and lipids with which it isnaturally associated to permit therapeutic administration. Such anantibody "preferentially binds" to an LCF polypeptide (or a fragment oranalog thereof), i.e., does not substantially recognize and bind toother antigenically-unrelated molecules.

Preferably, the antibody neutralizes the biological activity of theprotein to which it binds. By "neutralize" is meant to partially orcompletely block (e.g., the biological activity of a LCF polypeptide).

In other aspects, the polypeptides or antibodies described above areused as the active ingredient of therapeutic compositions. In suchtherapeutic compositions, the active ingredient is formulated with aphysiologically-acceptable carrier. These therapeutic compositions areused in a method of suppressing or mimicking LCF-CD4 interactionmediated physiological response. In particular, these methods are usedto reduce an immune response, or inflammation, or growth of an unwantedcell. Compounds useful in practicing the method include, withoutlimitation, an LCF antibody, or an LCF fragment or analog, or a drug,e.g. an organic compound.

In another aspect, the invention features an LCF immunoassay kitincluding an antibody of the invention. Preferably, such a kit includesa means for detecting the binding of the antibody to the LCFpolypeptide.

In another aspect, the invention features a method of detecting LCF in abiological sample, the method involving (a) contacting the biologicalsample with an antibody of the invention; and (b) detecting immunecomplex formation between the antibody and a sample constituent asindicative of the presence of LCF in the sample. Preferably, the methodinvolves an immune complex formation which is detected by an ELISA or aWestern blot analysis.

In yet another aspect, the invention features a method of screeningcandidate compounds for their ability to inhibit interaction between LCFand CD4. The method involves: a) mixing a candidate antagonist compoundwith LCF; b) measuring LCF-CD4 binding; and c) identifying antagonisticcompounds as those that interfere with the binding.

In still another aspect, the invention features a method of screeningcandidate compounds for the ability to mimick LCF activity, the methodinvolving: a) mixing a candidate agonist compound with CD4 receptor; b)measuring binding of the compound to CD4 receptor; and c) identifyingagonist compounds as those that bind CD4 receptor and mediate cellmigration.

In another aspect, the invention features a composition for stimulatingproliferation of CD4+ T-cells in a mammal, the composition including LCFand a growth factor. In preferred embodiments, the composition includesLCF and a growth factor in a ratio which causes synergy, e.g., rangingfrom 1:100 to 1:1 (LCF to growth factor). Preferably, the growth factoris a cytokine e.g., IL-2, IL-4, IL-6, IL-7, IL-8, insulin, andinsulin-like growth factor I.

The invention also features a method for stimulating proliferation ofCD4+ T cells in a mammal, the method includes contacting cells with LCFand IL-2 together or close enough in time to cause synergy. In preferredembodiments, the method includes administering to a mammal (e.g., ahuman patient) an effective amount of LCF and a growth factor, whereinthe proliferative activity of LCF in combination with the growth factoris greater than the prolifgrative activity of the LCF in the absence ofthe growth factor and the proliferative activity of the growth factor inthe absence of LCF. In preferred embodiments the growth factor is acytokine and, if desired, the administration of the composition occursmore than once.

In other preferred embodiments, the method for stimulating proliferationof CD4+ T cells involves (a) contacting cells with LCF and IL-2 in vitroand returning the proliferated cells into the mammal. Preferably, thestimulated CD4+ T cell is a PBMC or a HIV+ PBMC. In other preferredembodiments, the method further involves contacting the cells with ananti-retroviral agent (e.g., AZT or ddI).

In another aspect, the invention features a method for stimulatingproliferation of CD4+ T cells in a human infected with HIV, involvingadministering an effective therapeutic amount of a composition includingLCF and a growth factor. In preferred embodiments, the infected human isan asymptomatic human infected with HIV. In still other preferredembodiments, the human infected with HIV has a CD4+ count greater than50.

In another aspect, the invention features a method for stimulatingproliferation of CD4+ in a human having an immune disorder, the methodinvolving administering an effective therapeutic amount of a compositionincluding LCF and a growth factor.

In another aspect, the invention features a method for inducing theproliferation of CD4+ T cells in a human, the method involvingadministering an effective therapeutic amount of a composition includingLCF.

In still another aspect, the invention features a method of inhibiting aCD4+ bearing malignant cell in a mammal, involving administering to themammal (e.g., a human patient), a therapeutically effective amount of anLCF anatgonist (as described herein). In preferred embodiments, the CD4+T cell is a lymphoma or is a leukemia. Preferably, the antagonist orinhibitor is a LCF fragment or analog thereof or is an anti-LCFantibody. In other preferred embodiments, the method further involvesadministering to the mammal a chemotherapeutic agent in an effectivedose which is lower than the standard dose when the chemotherapeuticagent is used alone.

In another aspect, the invention features a method of protecting amammal from developing a neoplasm, involving administering to the mammal(e.g., a human patient) a therapeutically effective amount of an LCFantagonist.

The proteins of the invention are involved in events leading to inducingthe migration of specialized immune cells, e.g., eosinophils, monocytes,and T lymphocytes, which are important constituents and mediators ofboth the immune response and inflammation. Such proteins are thereforeuseful to treat or, alternatively, to develop therapeutics to treathyperresponsive immune reactions and inflammation that pertain to theactivation and subsequent infiltration of T lymphocytes, monocytes andeosinophils. Particular disorders which may be treated using theproteins and/or the methods of the present invention include, withoutlimitation, any granulomatous immune reaction, e.g., as effected bytissue-invasive helminth parasites, cutaneous and respiratory late-phasereactions to allergens, asthma, sarcoidosis, hypersensitivitypneumonitis, interstitial pulmonary fibrosis, tuberculosis, rheumatoidarthritis, and lupus erythematous, allogenic organ transplant rejection,contact (cell-mediated) dermatitis, and immunologically mediated skindiseases (e.g. pemphigoid and bullous pemphigoid). A comprehensive texton the aforementioned disorders may be found in Principles of InternalMedicine 12th ed. (Wilson et al., McGraw Hill, Inc., N.Y.). Preferredtherapeutics include antagonists, e.g., peptide fragments, orantibodies, or drugs, which block LCF or LCF:CD4 receptor function byinterfering with the LCF:CD4 receptor interaction and any concomitantbiological activity directed by LCF. Similarly, the antibodies of theinvention are useful for detecting the presence and clinical course ofany disease associated with LCF, e.g., those diseases described above.

Recombinant LCF can also be used as an immunosuppressive agent or aspart of immunosuppressive therapy. In particular, recombinant LCF mayserve to attenuate, interrupt, or prevent the cascade of events thateventually result in immunological rejection of tissue or organtransplants. For example, recombinant LCF may be used to attenuate,interrupt, or prevent a patient from rejecting a kidney, lung, orcombined heart-lung, or liver transplants. Further, recombinant LCF byvirtue of its ability to interact and bind with CD4 receptors may beuseful in the design of immunotoxins that selectively destroy CD4+receptor bearing cells. Finally, recombinant LCF may be used, alone orin combination with other compounds (e.g. growth factors), to activateand replenish a CD4 lymphocyte population in any patient with a depletedpopulation.

Because LCF may now be produced by recombinant techniques, and becausecandidate antagonists or agonists may be screened according to theassays described herein, the instant invention provides a simple andrapid approach to the identification of useful therapeutics. Such anapproach was previously difficult because LCF was unavailable insufficient quantities to identify its role in disease in animal models,and antibodies and DNA and RNA probes were previously unavailable fordetection of LCF protein or gene expression in diseased tissues.

Thus, once identified, a peptide- or antibody-based therapeutic may beproduced, in large quantity and inexpensively, using recombinant andmolecular biological techniques, and the methods of the presentinvention. Finally, any chemical compound, e.g., an organic compound,may be easily screened according to the methods outlined herein in orderto evaluate their effect on LCF:CD4 interaction.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS DRAWINGS

The drawings will first be described.

FIG. 1 shows a northern analysis of LCF from total cellular RNA preparedfrom human T lymphocytes. Positions of 18S and 28S RNA visualized byethidium bromide staining are shown at their respective arrows.

FIG. 2 shows the nucleotide sequence of the LCF-A cDNA (SEQ ID NO: 2)and predicted amino acid sequence of the encoded protein (SEQ ID NO: 1).Nucleotides are numbered on the left side beginning with the firstnucleotide of the cDNA. The poly A tail begins immediately after thelast indicated nucleotide (2152) and is omitted. Translation of theputative LCF coding sequence is indicated below the correspondingnucleotide sequence starting with Met. Each amino acid is consecutivelynumbered. An Asn residue (amino acid residue 5) represents a potentialglycosylation site (marked with a dot). Two candidate polyadenylationsignal sequences are underlined.

FIG. 3A and FIG. 3B show a SDS-PAGE of recombinant LCF expressed in E.coli and a rabbit reticulocyte in vitro translation of RNA synthesizedfrom LCF cDNA. FIG. 3A shows recombinant LCF protein run on a 15%SDS-PAGE followed by coomassie blue staining. In FIG. 3A, lane A showscrude supernatant from E. coli induced to express LCF protein, lane Bshows LCF protein generated as a fusion protein conjugated to apolyhistidine linker purified by nickel affinity chromatography, andlane C shows LCF after Factor Xa cleavage. The band at 17.5 kDa wasblotted, excised and subjected to N-terminal amino acid sequencing. FIG.3B shows a rabbit reticulocyte in vitro translation of LCF cDNA: the ³⁵S-labeled protein product of LCF cDNA translated by rabbit reticulocyteswas run on a 15% SDS-PAGE. In FIG. 3B, lane A shows LCF proteintranslated under non-glycosylating conditions, and lane B shows LCFtranslated under glycosylating conditions.

FIG. 4 shows the immunoprecipitation of recombinant LCF by rsCD4. InFIG. 4, lane 1 shows 10 μg of recombinant LCF; lane 2 shows recombinantLCF incubated with 50 μg rsCD4 immunoprecipitated with 10 μg rabbitpolyclonal anti-CD4 antibody; lane 3 shows recombinant LCF incubatedwith 10 μg rsCD4 immunoprecipitated with polyclonal anti-CD4 antibody;lane 4 shows recombinant LCF incubated with rsCD4 (10 μg)immunoprecipitated with rabbit polyclonal anti-IgG (10 μg); lane 5,shows recombinant LCF incubated with rsCD4 and immunoprecipitated withmonoclonal anti-CD4 (10 μg); lane 6, shows recombinant LCF incubatedwith rsCD4 and immunoprecipitated with monoclonal anti-CD8 antibody (10μg); and lane 7, shows rsCD4 (10 μg) incubated with monoclonal anti-CD8antibody.

FIG. 5 shows a dose response curve for recombinant LCF inducedchemotaxis of human peripheral blood T lymphocytes. In FIG. 5, anasterisk (*) represents statistical significance at p<0.05 (using aStudent's T test from control cell migration).

FIG. 6 shows recombinant LCF-induced chemotaxis in murine T cellhybridoma cells. Murine cell lines expressing either wild-type CD4(13.13), truncated CD4 (delta-13), or mock infected cells lacking CD4expression (155.16) were stimulated by recombinant LCF (10⁻⁹ M) (openbars) or 2C11 antibody (10 μg/ml) (striped horizontal bars) and themigratory response quantitated. Cells stimulated by recombinant LCF inthe presence of a 100 fold excess of anti-CD4 Fab fragments (10 μ/ml)are also shown (solid bars). Cell migration is expressed as mean of tenhigh power fields ±S.D. Migration which was significantly different(p<0.05 by Student's T test) from control cell migration (designated as100%) is indicated by asterisks.

FIG. 7 shows the specificity of recombinant LCF for CD4+ human T cellsusing FACs analysis. Two×10⁶ human T lymphocytes were cultured for 24and 48 h in the presence of 10⁻⁸ M recombinant LCF. Cellsdouble-labelled with phycoerythrin-conjugated anti-CD4 antibody andfluorescein-conjugated anti-IL-2R antibody were analyzed on a BectonDickinson FACscan flow cytometer. Recombinant LCF induced an increase inCD4+/IL-2R⁺ cells from a control level of 3% (top panel) to 17% (bottompanel) by 48 h. The 24 h time point demonstrated an increase in 9% ofthe cells. At no time did CD4⁻ cells show an increase in IL-2Rexpression. This is a representative FACs analysis of three differentexperiments. Other experiments demonstrated increases in IL-2R⁺ cells atthe 48 h time point in 15% and 19% of the cells.

FIG. 8A and FIG. 8B show the aggregation of recombinant LCF underphysiological conditions. FIG. 8A shows a molecular sieve HPLC of ³⁵S-labelled recombinant LCF (run in phosphate buffered saline, pH 8.0).Fractions were collected and analyzed by scintillation counting (opensquares). Parallel samples were collected and assayed for the inductionof lymphocyte chemotaxis (solid squares). FIG. 8B, lane A and lane Bshow an autoradiogram of the peak fraction for both radioactivity andcell migration (fraction 13 shown in FIG. 8A) and the second peak ofradioactivity which had no corresponding chemoattractant activity(fraction 17 shown in FIG. 8A) after separation by SDS-PAGE,respectively.

FIG. 9 shows a hydrophilicity plot of recombinant LCF predicted by themethod of Kyte and Doolittle (Kyte et al., J. Molec. Bio. 157:105-132(1982)). Peptides were synthesized and rabbit anti-peptide specificanti-sera were generated to four major hydrophilic regions designated byA,B,C,D.

FIG. 10 shows induced chemotaxis of human T lymphocytes by concentratedBAL fluid from normal individuals. Fifty milliliters of BAL fluid wasconcentrated 100 fold and then assayed diluted 1:1 in phosphate bufferedsaline in a microchemotaxis chamber. The data is expressed as a percentof random cell migration in the presence of PBS alone (normalized to100% in all experiments, for these experiments control migrationaveraged 14.3 cells/hpf). Each BAL fluid was assayed three times, withthe asterisks denoting migration statistically different from controlcell migration (p<0.05).

FIG. 11 shows the induced migration of peripheral T cells byconcentrated BAL fluids from asthmatics following either saline (solidbars) or specific antigen (hatched bars) challenge. The BAL fluids wereobtained 6 hrs after challenge and concentrated 100 fold prior toassaying. Each BAL fluid was assayed three times with the asterisksdenoting cell migration which was statistically different from controlcell migration (p<0.05). For these experiments control migrationaveraged 12.5 cells/hpf.

FIG. 12A and B, show the blocking effect of a panel of anti-cytokineantibodies on the induction of peripheral T cell migration by BALfluids. Positive BAL samples, as determined in FIG. 11, were reassessedfor the induction of T cell migration (as shown in panel a) either alone(solid bars ), in the presence of anti-LCF polyclonal antibody (shadedbars), or with anti-MIP1α polyclonal antibody (left hatched bars). Panel(b) shows BALs alone (solid bars), with anti-IL-8 polyclonal antibody(stippled bars), or anti-RANTES monoclonal antibody (horizontal bars).All antibodies were used at a concentration sufficient to neutralizebioactivity from 50 ng/ml of protein. The experiment was conducted threedifferent times and the asterisks denotes cell migration statisticallydifferent from cell migration induced by the same BAL sample assayedalone (p<0.05). Control migration in these experiments averaged 15cells/hpf.

FIG. 13 shows the blocking of BAL fluid-induced T cell migration byanti-LCF, anti-MIP1α or a combination of the two antibodies. Inductionof cell migation was assessed for BAL samples incubated either alone(solid bars), in the presence of anti-MIP1α antibody (left hatchedbars), in the presence of anti-LCF antibody (shaded bars), or in acombination of the two antibodies (horizontal bars). Antibodies wereused at concentrations sufficient to neutralize bioactivity from 50ng/ml of specific protein. The data is expressed as percent of controlcell migration, with asterisks denoting inhibition of migration whichwas statistically different from BAL-induced cell migration in theabsence of blocking antibodies (p<0.05). Control migration in theseexperiments averaged 13.8 cells/hpf.

FIG. 14 shows the effect of media, rIL-2, rLCF, and rLCF and IL-2 onhuman nylon wool non-adherent T cells (NWNAT) proliferation.

FIG. 15 shows the effect of media, rIL-2, rLCF, and rLCF and IL-2 onhuman HIV+ PMBCs.

FIG. 16 shows the cell counts of long term cultures of rLCF and rIL-2treatments with CD4 counts obtained from patients infected with HIV.Data for 5 and 6 represent the same individual a month apart.

FIG. 17 shows p24 measurement by ELISA.

LCF POLYPEPTIDES

LCF polypeptides according to the invention include the full-length LCFpolypeptide (as described in FIG. 2, SEQ ID NO: 1). Such polypeptidesmay be derived from any source. These polypeptides are used, e.g, toscreen for antagonists which disrupt a LCF:CD4 receptor interaction oran LCF:mediated physiological response (see below). LCF fragments oranalogs may also be useful candidate antagonists of LCF:CD4 receptoractivity. The efficacy of a LCF fragment or analog antagonist isdependent upon its ability to interact with CD4; such an interaction maybe readily assayed using any number of standard binding methods andLCF-mediated CD4 receptor functional assays (e.g., those describedbelow). Polypeptides of the invention also include any fragment oranalog capable of interacting with the CD4 receptor and mediating theLCF biological cascade, i.e. LCF agonists.

Specific LCF polypeptide fragments of interest include any portion ofthe LCF polypeptide which are capable of interaction with CD4 receptor,e.g., all or part of the N-terminus or e.g., a hydrophilic domain. Basedon the hydrophilicity analysis (see FIG. 9) and biologic inhibitiondata, other likely candidates include without limitation, the fourhydrophilic regions, A, B, C and D (see FIG. 5) and the FEAW (Phe, Glu,Ala, Trp) sequence from amino acids 96-99 of LCF (FIG. 2 and SEQ ID NO:1). Such fragments may be useful as agonists or antagonists (asdescribed above), and are also useful as immunogens for producingantibodies which neutralize the activity of LCF; see infra).

Alternatively, from the primary amino acid sequence the secondaryprotein structure and, therefore, the domains of LCF may be deducedsemi-empirically using any standard hydrophobicity/hydrophilicitycalculation, e.g., the Chou-Fasman method (see,e.g., Chou and Fasman,Ann. Rev. Biochem. 47:251, 1978). Hydrophilic domains present themselvesas strong candidates for antigenicity and hydrophobic regions forbinding domains, and therefore, useful antagonists or agonists.

Candidate fragments (e.g., all or part of Domains A or D; see, FIG. 9)are then tested for interaction with CD4 receptor and their ability toinduce an LCF-mediated physiological response, i.e., serve as LCFagonists, by assays described herein. Fragments are also tested fortheir ability to antagonize the interaction between LCF and CD4 usingthe assays described herein. Analogs of useful LCF fragments (asdescribed above) may also be produced and tested for efficacy asscreening components or antagonists (using the assays described herein);such analogs are also considered to be useful in the invention.

There now follows a description of the cloning and characterization of ahuman LCF cDNA useful in the instant invention. This example is providedfor the purpose of illustrating the invention, and should not beconstrued as limiting.

Isolation of Human LCF cDNA

The human LCF gene was isolated as follows.

A cDNA library from mitogen-stimulated human peripheral bloodmononuclear cells (PBMC) was ligated into the COS cell expression vectorpXM (Wong et al., Science 228:801-815, 1985). Supernatants from cellstransfected with pooled plasmids were screened for lymphocytechemoattractant activity using a modified Boyden chamber assay(Cruikshank et al., J. Immunol. 128:2569-2574, 1982). Supernatantscollected 24 h after transfection were placed in bottom wells ofmicrochambers. The migration of human T cells through 8 μmnitrocellulose filters in response to the presence of these supernatantswas determined, compared to supernatant of mock (vector only)transfected COS cells. Supernatants with chemoattractant activity werefurther screened for the capacity to induce IL-2R expression on restingT-cells by FACS analysis of cells incubated with fluorescein-conjugatedanti-Tac antibody, and for the ability of Fab fragments of monoclonalOKT4 antibody to block this induction (Cruikshank et al., J. Immunol.138:3817-3723, 1987). Seven different subclonings were screened,approximately 200 clones per supernatant in original supernatants thatwere subcloned were found to be positive. Next, the supernatants weresequentially subcloned and diluted until one clone per supernatant wasobtained. The criteria established for the presence of LCF-containingsupernatant included a positive response for both assays and, inaddition, that the activity could be blocked by coincubation with Fabfragments generated from OKT4 antibodies (Ortho Pharmac, Raritan, N.J.).A single clone (LCF-7) with these characteristics was isolated and bothstrands were sequenced by the dideoxynucleotide chain termination method(Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977). Sequenceanalysis and northern blotting (FIG. 1) indicated that the LCF-7 cDNAwas not full-length (corresponding to nucleotide 441 to 1450 of theindicated sequence). Then, the LCF-7 cDNA was used to probe a secondmitogen-stimulated human PBMC cDNA library ligated into bacteriophagelambda ZAP. 125,000 plaques were screened with full length LCF-7. Uponscreening, three clones were isolated ranging in size from 0.6- to2.2-kb. The largest clone was sequenced on both strands (see FIG. 2; SEQID NO: 2). Partial sequencing of two shorter clones revealed that theywere identical to LCF-A, but incompletely extended in the 5' direction.

As described above, LCF cDNA was isolated by screening a COS cellexpression library of mitogen-stimulated human peripheral bloodmononuclear cells (PMBC). Supernatants were assessed for the presence ofLCF by the induction of human CD4+ T cell chemotaxis and cell cyclechanges as determined by upregulations of IL-2 receptors (IL-2R)(Cruikshank et al., J. Immunol. 138:3817-3823, 1987). Following fourrounds of screening, a positive supernatant from a single clone of 1-kbwas identified. The LCF cDNA was used to probe a northern blot of totalRNA isolated from human T cells (FIG. 1). A single band of 2.2-kb wasdetected. In order to isolate a full length clone the 1-kb LCF cDNA wasused to probe a second mitogen-stimulated human PBMC cDNA library. Threeclones were isolated, and the sequence of the largest clone is shown inFIG. 2 and SEQ ID NO: 2.

Within the LCF cDNA there is an open reading frame of 393 base pairsextending from nucleotide 783 to 1176 that codes for a 130 residueprotein with a predicted molecular mass of 13,385 daltons. Themethionine at nucleotide 783 is in good context for initiation byFickett analysis (Fickett, Nucleic Acids Res. 10:5303-5318, 1982). Theonly other possible initiation site lies downstream and is in-frame,representing residue 38 of the predicted amino acid sequence. There isone potential N-linked glycosylation site on the serine located fiveresidues after the start methionine. While previous work suggests thatnative LCF is a secreted cytokine (Cruikshank et al., J. Immunol.128:2569-2574, 1982), in the predicted amino acid sequence there is noconsensus hydrophobic signal sequence; however, nor is there a potentialtransmembrane domain. While most secreted cytokines contain a signalsequence, the absence of a signal sequence has been reported for bothsecreted IL-1α and IL-1β. Similarly searches of the Genbank nucleic acidand protein data bases failed to find any related sequences. DNA andprotein homology searches were conducted using the programs FASTA,SEARCH, and BLAST in the Genbank and PIR databases.

RNA Isolation and Northern Analysis

Human peripheral blood mononuclear cells (PBMC) were prepared byFicoll-Paque density centrifugation as previously described (Cruikshanket al., J. Immunol. 138:3817-3823, 1987; Cruikshank et al., J. Immunol.146:2928-2934, 1991). The T lymphocyte population was purified byplastic adherence followed by nylon wool adherence and finally by sheeperythrocyte rosetting and centrifugation. Cells recovered from thepellet were >99% T lymphocytes as determined by fluorescent analysis.Monocytes were purified from PBMC using sheep erythrocyte resetting todeplete T lymphocytes, followed by plastic adherence of the cellsremaining in the supernatant after the rosetting step. Adherent cellsrecovered from the plastic were >92% monocytes by fluorescence analysis.All cells were lysed with cold 4M guanidinium isothiocyanate and RNA wasisolated by CsCl centrifugation (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1989). Ten μg of RNAfrom each sample was loaded on a 1% agarose-formaldehyde gel forelectrophoresis, and blotted onto nylon membrane. A cDNA probe from a704 bp Pst I fragment of recombinant LCF-7 was ³² P!dCTP-labeled by therandom primer method (Feinberg et al., Anal. Biochem. 132:6-13, 1983)and the blot was hybridized with 1×10⁶ cpm/ml for 24 hr. Afterhybridization the blot was washed with 0.2×SSC (30 mM NaCl, 3 mM sodiumcitrate, 0.05% sodium pyrophosphate, 0.1% sodium lauryl sarcosine) at56° C., and hybridization was visualized by autoradiography. As shown inFIG. 1, the probe hybridized specifically to a lymphocyte RNA ofapproximately 2.2 kilobases. This confirmed that LCF was expressed in Tlymphocytes and indicated that the clone was full-length.

LCF Polypeptide Expression and Synthesis

Polypeptides according to the invention may be produced bytransformation of a suitable host cell with all or part of anLCF-encoding cDNA fragment (e.g., the cDNA described above) in asuitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant LCF protein. The precise host cell used is not critical tothe invention. The LCF polypeptide may be produced in a prokaryotic host(e.g., E. coli), or in a eukaryotic host (e.g., S. cerevisiae ormammalian cells, e.g., COS1, NIH3T3, and JEG3 cells, or in the cells ofan arthropod, e.g. Spodoptera frugiperda (SF9) cells). Such cells areavailable from a wide range of sources (e.g., the American Type CultureCollection, Rockland, Md.; also see, e.g., Ausubel et al., supra). Themethod of transfection and the choice of expression vehicle will dependon the host system selected. Transformation and transfection methods aredescribed, e.g., in Ausubel et al., supra; expression vehicles may bechosen from those provided, e.g., in Cloning Vectors: A LaboratoryManual (P. H. Pouwels et al., 1985, Supp. 1987).

One preferred LCF expression system is a prokaryotic expression systemas described by Ausubel et al. (supra). Thus, a DNA fragment containingthe LCF cDNA open reading frame with flanking BamH1 and Nde1 restrictionsites was generated by PCR according to standard methods and ligatedinto the E. coli expression vector pT-16b (Novagen). This plasmid,pET-166-ICF, was then used to transform E. coli JM109. In order tostimulate the production of recombinant LCF the transformed bacterialwere stimulated with IPTG, grown in culture media and subsequentlylysed. Recombinant protein was isolated by metal chelationchromatography according the well known methods (see, e.g., StudierMeth. Enzymol. 185:60-89, 1990). Recombinant LCF was then subjected toSDS-PAGE (FIG. 3A) and blotted to Problott transfer filters (AppliedBiosystems). A prominent band found at an apparent molecular weight of17.5 kDa was excised and subjected to N-terminal amino acid sequencingaccording to standard techniques. Twenty-five amino acid residues at theN-terminus of the recombinant LCF were sequenced and were found to beidentical to the predicted amino acid sequence shown in FIG. 2 (SEQ IDNO: 1). While the SDS-PAGE mass of 17.5 kDa is larger than the expected13.4 kDa based on nucleotide sequence, it is identical to the migrationpattern of ³⁵ S-labeled in vitro translated protein (FIG. 3B). Thediscrepancy in mass determined by SDS-PAGE from the predicted sequencemay be due to aberrant migration of recombinant LCF in the SDSacrylamide gel system.

Another preferred LCF expression system is a baculovirus expressionsystem as described by Ausubel et al. (supra). DNA encoding an LCFpolypeptide is inserted into an appropriate transfer vector, e.g.,pVL1392 (Invitrogen Corp., San Diego, Calif.). Next, the vector isco-transfected with wild type baculovirus genomic DNA into Spodopterafrugiperda (SF9) cells (ATTC Accession No: CRL 1711) and recombinantviruses are isolated by standard techniques, e.g., see Ausubel et al.(supra). Recombinant LCF produced in a baculovirus system was found tosynthesize a protein with an apparent molecular weight of 17.5 kDa whichis similar to the protein synthesized using the E. coli expressionsystem shown in FIG. 3A and FIG. 3B. Sequencing of the first fiveN-terminal amino acid residues of the baculovirus recombinant LCF wasperformed. The sequences were found to be identical to the predictedamino acid sequence shown in FIG. 2 (SEQ. ID No.: 1) with a methionineat position 783 as the initiation site.

Alternatively, an LCF polypeptide may be produced by astably-transfected mammalian cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public,e.g., see Pouwels et al. (supra); methods for constructing such celllines are also publicly available, e.g., see Ausubel et al. (supra). Inone example, cDNA encoding the LCF polypeptide is cloned into anexpression vector which includes the dihydrofolate reductase (DHFR)gene. Integration of the plasmid and, therefore, the LCF-encoding geneinto the host cell chromosome is selected for by inclusion of 0.01-300μM methotrexate in the cell culture medium (as described in Ausubel etal., supra). This dominant selection can be accomplished in most celltypes. Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are described in Ausubel et al. (supra);such methods generally involve extended culture in medium containinggradually increasing levels of methotrexate. DHFR-containing expressionvectors commonly used for this purpose include pCVSEII-DHRF andpAdD26SV(A) as described in Ausubel et al. (supra). Any of the hostcells described above or, preferably, a DHFR-deficient CHO cell line(e.g., CHO DHFR-cells, ATCC Accession No. CRL 9096) are among the hostcells preferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

Once the recombinant LCF polypeptide is expressed, it is isolated, e.g.,by using affinity chromatography. In a working example, a CD4 affinitycolumn was prepared by coupling recombinant soluble CD4 (rsCD4) to CNBrSepharose 4B according to previously described methods (see, e.g.,Cruikshank et al., Journal of Immunology 1991). Thus, 100 μg rsCD4 wascovalently conjugated to a CNBr activated Sepharose 4B (Pharmacia,Piscataway, N.J.). Next, an in vitro RNA transcript of LCF was generatedand used for in vitro translation with rabbit reticulocyte lysate in thepresence of ³⁵ S! methionine according to standard methods. ³⁵ S-labeledin vitro LCF was applied to the column for 3 hr at 37° C. at which timethe column was extensively washed with wash buffer (0.01M Tris-Cl, pH8.0, 0.14M NaCl, 0.025% NaN₃, 0.5% Triton X-100, 0.5% sodiumdeoxycholate). LCF was eluted with a triethanolamine solution (50 mMtriethanolamine, pH 11, 0.1% Triton X-100, 0.15M NaCl) into tubescontaining 1M Tris-Cl, pH 6.7 and analyzed.

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography. These generaltechniques of polypeptide expression and purification can also be usedto produce and isolate useful LCF fragments or analogs (as describedbelow). Furthermore, the eluate may then, if desired, be run on aSDS-PAGE and visualized by autoradiography (see, e.g., the results fromthe above experiment presented in FIG. 3B).

Finally, LCF polypeptides, particularly short LCF fragments, can beproduced by chemical synthesis (e.g., by the method described in SolidPhase Peptide Synthesis, 1984, 2nd ed. , Stewart and Young, eds., PierceChemical Co., Rockford, Ill.).

Assays for LCF Binding and Function

Useful LCF polypeptide fragments or analogs in the invention are thosewhich interact with CD4 receptor, e.g., LCF agonists or antagonists.Such an interaction may be detected by an in vitro binding assay (asdescribed infra) followed by functional analysis. Thus, the fragments oranalogs thereof may also be assayed functionally, i.e., for its abilityto bind a CD4 receptor and to induce the migration of T4+ lymphocytes,monocytes, eosinophils and the like (as described infra). These assaysinclude, as components, LCF (or a suitable LCF fragment or analogthereof) and recombinant soluble CD4 receptor (rsCD4) or CD4receptor-bearing cell, e.g., an eosinophil, configured to permitdetection of binding. Thus, the invention includes methods for screeningcompounds useful as LCF agonists.

One such assay method is as follows. Full-length LCF polypeptide(fragment or analog thereof) is produced as described supra. CD4receptor component is produced either as a recombinant soluble componentor is produced as a membrane component by a cell, e.g., a T lymphocyte,monocyte or eosinophil.

In vitro assays to determine the extent of LCF (fragment or analogthereof) binding to rsCD4 or CD4 receptor-bearing cells is thenperformed. For example, a whole cell assay is preferably performed byfixing the cell expressing the CD4 receptor, e.g, eosinophils, to asolid substrate (e.g., a test tube, or a microtiter well) by means wellknown to those in the art (see, e.g., Ausubel et al. supra) andpresenting labelled LCF polypeptide (e.g., ¹²⁵ I-labelled LCF).Labelling of LCF, e.g., with ¹²⁵ I, is performed according to standardtechniques known in the art. Binding is assayed by the detection labelin association with the receptor component (and, therefore, inassociation with the solid substrate and CD4 receptor) by techniqueswell known in the art.

The assay format may be any of a number of suitable formats fordetecting suitable binding, such as a radioimmunoassay format (see,e.g., Ausubel et al., supra). Preferably, cells bearing CD4 receptor areimmobilized on a solid substrate (e.g., the well of a microtiter plate)and reacted with LCF polypeptide which is detectably labelled, e.g.,with a radiolabel such as ¹²⁵ I or an enzyme which can be assayed, e.g.,alkaline phosphatase or horseradish peroxidase. Thus, ¹²⁵ I-labelled LCFis bound to the cells and assayed for specific activity; specificbinding is determined by comparison with binding assays performed in thepresence of excess unlabelled LCF polypeptide.

Alternatively, LCF polypeptide (fragment or analog thereof) may beadhered to the solid substrate (e.g., to a microtiter plate usingmethods similar to those for adhering cells for an ELISA assay; Ausubelet al. supra) and the ability of labelled rsCD4 receptor to bind LCF canbe used to detect specific rsCD4 receptor binding to the immobilizedLCF.

There now follows an example demonstrating still another method usefulfor analyzing the LCF:CD4 interaction. In this method recombinantLCF-containing E. coli crude supernatant was incubated with 10 μg ofrsCD4 for 1 h at 4° C. Next, the recombinant LCF-CD4 complex was addedto protein A Sepharose beads which had been incubated with 1 μg rabbitanti-CD4 polyclonal antibody and washed with a suitable buffer. Themixture was then incubated for 2 h at 4° C., washed four times with TSB(0.01M Tris, (pH 8.0), 0.14M NaCl, 0.025% NaN₃) prior to running on a15% SDS-polyacrylamide gel system. Protein separated on the SDS-gel wasthen transferred to Problott transfer filters and probed using rabbitanti-peptide D antibody (1:200 dilution) (also see section infraanti-LCF Antibodies) followed by goat anti-rabbit ¹²⁵ I-IgG antibody.The results of this experiment are presented in FIG. 4. As shown in FIG.4, there is a detectable specific physical interaction betweenrecombinant LCF and rsCD4.

LCF polypeptide (or fragment or analog thereof) may also be assayedfunctionally for its ability to mediate migration of CD4+ lymphocytes,monocytes, eosinophils and the like. Migration assays may be employedusing any suitable cell, e.g., T lymphocytes, monocytes or eosinophilsas described in (Cruikshank et al., 1987, J. Immunol. 128:2569-2571;Rand et al., 1992, J. Exp. Med. 173:1521-1528) follows. For example,recombinant LCF synthesized in an expression system, e.g., E. coli orbaculovirus expression systems (as described supra), can be assayed forthe ability to induce cell migration. In one working example, murinecell chemotaxis was performed using a modified Boyden chemotaxis chamber(Cruikshank et al, J. Immunol. 128:2569-2571). The cells were suspendedin RPMI 1640 containing 10% FBS at a concentration of 5×10⁶ cells/ml. A12 μm nitrocellulose membrane was used and the cells were incubated for4 h. Next, the membranes were stained with hematoxylin and dehydratedusing sequential washing with ethanol, propanol, and finally xylene toclarify the filters and allow for cell counting by light microscopy.Cell migration was quantitated by counting the number of cells which hadmigrated beyond 50 μm. All counts were compared with control cell(unstimulated) migration which was always normalized to 100%. Inaddition, all samples were performed in duplicate and five high-poweredfields were counted for each duplicate. FIG. 5 shows a representativedose response curve for protein generated from the E. coli expressionsystem (supra). As indicated from the dose response curve, maximalmigration was induced with a concentration of recombinant LCF at 10⁻⁹ M,and ED₅₀ of 10⁻¹¹ M. Statistics were performed using Student's T Test(or analysis of variance modifications when data from multipleexperiments were pooled) and counts statistically different from controlcell migration (p<0.05) are designated by an asterisk. Similar resultswere obtained when baculovirus-produced LCF was substituted for E.coli-produced LCF.

In order to demonstrate that this physical association betweenrecombinant proteins in solution corresponds to a specific functionalassociation between recombinant LCF and cell surface CD4 the effects ofrecombinant LCF on murine T cell hybridoma cell lines expressing eitherfull-length or truncated human CD4 was examined (Sleckman et al., 1987,1988). Three cell lines were employed: a mock infected cell line whichlacked expression of CD4; a cell line expressing intact (wild type) CD4;and a cell line expressing truncated CD4 (delta 13) in which the 31 mostdistal residues of the cytoplasmic tail of CD4 have been deleted. Thecell lines expressing either intact CD4 or delta 13 CD4 were chosen fortheir comparable levels of CD4. As shown in FIG. 6 cells which expressedintact CD4 migrated in response to recombinant LCF stimulation. Cellseither lacking CD4 or expressing delta 13 CD4 were unresponsive torecombinant LCF. These cells were responsive to murine T cellreceptor-stimulated migration as the antibody 2C11 induced migratoryresponses of 198% ±4% and 192% ±3% for the mock transfected and delta 13CD4 cell lines respectively (FIG. 6). These studies demonstrate that CD4must be expressed for LCF-induced cell motile responsiveness and thatthe cytoplasmic tail is required.

CD4 specificity for LCF stimulation in human T cells was demonstratedusing the expression of IL-2R to identify LCF responsive cells. Mixed Tcells were cultured in the presence of recombinant LCF (10⁻⁸ M) for 24and 48 hrs at which time the cells were labeled for their expression ofboth CD4 and IL-2R. As shown in FIG. 7, only cells which were CD4⁺demonstrated an increase in surface expressed IL-2R. In this particularexperiment an increase in IL-2R was observed for 17% of the CD4⁺ cells.This indicates not only LCF specificity for CD4⁺ cells, but alsosuggests that recombinant LCF acts only on a subset of CD4⁺ cells.

Finally, molecular weight sieve chromatography of recombinant LCF showsthat most chemoattractant activity elutes in the 50-60 kDa region. Thispeak of chemoattractant activity corresponds to the elution profile of³⁵ S-labeled recombinant LCF subjected to identical chromatography asshown in FIG. 8A and FIG. 8B. A small peak of radioactivity was presentwith no corresponding chemoactivity in the 14-18 kDa region. The peakfraction for both chemotaxis and radioactivity (fraction 13) and thefraction containing only radioactivity (fraction 17) were applied toSDS-PAGE and subjected to autoradiography. The LCF proteins from eachfraction appeared as single bands at 17.5 kDa (FIG. 8B). These datasuggest that under physiologic conditions LCF exists predominantly as anon-covalently linked multimer, but some LCF may exist as monomers. Themultimeric form, however, is believed to possess chemoattractantactivity.

Screening For Compounds that Inhibit LCF:CD4 Interaction

As discussed above, one aspect of the invention features screening forcompounds that antagonize the interaction between LCF and CD4 receptor,thereby preventing or reducing the cascade of events that are mediatedby that interaction. Chemical antagonists to LCF which bind to LCF orLCF/CD4 receptor or CD4 receptor without triggering a response are usedto reduce, attenuate or interfere with the effects of LCF orcross-linked LCF agonists or biologically active LCF polypeptidefragments or analogs thereof which act to stimulate or activateLCF-mediated events of the immune response and inflammation. Thus, theinvention provides for methods to screen for such useful compounds.These antagonists include, without limitation, e.g., cross-linked LCF,synthetic LCF, anti-LCF antibodies, or other drugs, e.g. organiccompounds.

Thus, LCF polypeptide can be used to prepare compounds that tend toneutralize or impede its activity. For example, one approach pertains toidentification of the active sites of LCF, followed by the alteration ofthose sites of the LCF amino acid sequence by substitution of aminoacids within the active site by other amino acids, so that the peptidedoes not lose its binding affinity for the CD4 receptor, but uponbinding is unable to promote activity, and thereby blocks the effect ofLCF. LCF activity may also be blocked, attenuated, or interfered with byusing antibodies, e.g., monoclonal, or chemical antagonists to LCF.These chemical antagonists include any organic compounds, or any of theother aforementioned compounds, which can be assayed or screened fortheir ability to interfere with LCF:CD4 mediate events by the methodsthat follow.

The elements of the screen are LCF polypeptide (or a suitable fragmentor analog thereof) and rsCD4 or, a CD4 receptor expressing cell, e.g.,CD4⁺ lymphocyte, monocyte, eosinophil and the like, configured to permitdetection of binding. A full-length LCF polypeptide (fragment or analogthereof) and rsCD4 may be produced as described above.

Binding of LCF to its receptor may be assayed by any suitable method (asdescribed above). For example, cells expressing CD4 receptor, e.g.,eosinophils, are immobilized on a solid substrate (e.g., the well of amicrotiter plate) and reacted with detectably-labelled LCF polypeptide(fragment or analog thereof) as described above. Binding is assayed bythe detection label in association with the receptor component (and,therefore, in association with the solid substrate). Binding of labelledfull-length recombinant LCF polypeptide to CD4 receptor bearing cells isused as a "control" against which antagonist assays are measured. Theantagonist assays involve incubation of the CD4 receptor bearing cellswith an appropriate amount of candidate antagonist, e.g., an antibody oran organic compound. To this mix, an equivalent amount of labelled LCFis added. An antagonist useful in the invention interferes withlabelled-LCF binding to the immobilized receptor-bearing cells.Alternatively, an antagonist may bind but not activate a biologicalresponse.

Subsequently, an antagonist, if desired, may be tested for its abilityto interfere with LCF function, i.e., to specifically interfere withlabelled LCF binding without resulting in signal transduction normallymediated by a full-length LCF polypeptide.

Appropriate candidate antagonists include e.g., the polypeptides FEAW(Phe-Glu-Ala-Trp at amino acids 96-99) and RKSLQSKETTAAGDS(Arg-Lys-Ser-Leu-Gln-Ser-Lys-Glu-Thr-Thr-Ala-Ala-Gly-Asp-Ser at aminoacids 116-130) see e.g., SEQ ID No.:1 analogs of LCF, and other peptidesas well as non-peptide compounds, and anti-LCF polypeptide antibodiesdesigned or derived from analysis of LCF/CD4 receptor interaction or theprimary structure of LCF.

Anti-LCF Polypeptide Antibodies

Human LCF (or fragments or analogs) may be used to raise antibodiesuseful in the invention; such polypeptides may be produced byrecombinant or peptide synthetic techniques (see, e.g., Solid PhasePeptide Synthesis, supra; Ausubel et al., supra). The peptides may becoupled to a carrier protein such as KLH as described in Ausubel et al.,supra. The KLH-peptide is mixed with Freund's adjuvant and injected intoguinea pigs, rats, donkeys and like or preferably rabbits. Antibodiesmay be purified by peptide antigen affinity chromatography.

For example, Kyte-Doolittle analysis (Kyte, J. Molec. Bio. 157:105-132,1982) of the predicted amino acid sequence revealed four majorhydrophilic regions (FIG. 9). Based on the LCF hydrophilicity plot,rabbit antibodies to synthetic polypeptides of the four majorhydrophilic regions from residues 3-11, 47-58, 68-81 and 115-130(designated in FIG. 9 as A, B, C, D, respectively) were generated.Peptide specific polyclonal antisera were identified by ELISA for eachpeptide and then purified by protein A sepharose chromatography. In oneexample demonstrating the utility of such antibodies, it was determinedthat antibodies generated to region D blocked recombinant LCF (10⁻⁹M)-induced migation from 194% ±7% (mean ±S.D., N=4) to 112% ±5% in thechemotaxis indicator assay system (described supra). Furthermore, theanti-peptide D antibody was found to be suitable for western blottingand identified the same 17.5 kDa band as was observed following proteinstaining in FIG. 3A and FIG. 3B.

Alternatively, monoclonal antibodies may be prepared using LCFpolypeptides described above and standard hybridoma technology (see,e.g. Kohler et al., Nature, 256:495, 1975; Kohler et al., Eur. J.Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol., 6:292, 1976;Hammerling et al., In Monoclonal Antibodies and T CellHybridomas,Elsevier, N.Y., 1981; Ausubel et al., supra). Thus, in oneexample, monoclonal antibodies to LCF (fragments or analogs thereof) canbe raised in Balb/C or other similar strains of mice by immunizationwith purified or partially purified preparations of LCF (fragments oranalogs thereof). The spleens of these mice can be removed, and theirlymphocytes fused to a mouse myeloma cell line. After screening ofhybrids by known techniques, a stable hybrid will be isolated thatproduces antibodies against LCF (fragments or analogs thereof). Suchactivity can be demonstrated by the ability of the antibody to preventthe binding of radio-labelled LCF (e.g., ¹²⁵ I-LCF) to the CD4 receptor.The monoclonal antibody can then be examined for its ability to preventthe biological activity of LCF, e.g., cell migration (as discussedabove). Once produced, polyclonal or monoclonal antibodies are testedfor specific LCF polypeptide recognition by Western blot orimmunoprecipitation analysis (by methods described in Ausubel et al.,supra). Antibodies which specifically recognize an LCF polypeptide(fragment or analog thereof) are considered to be likely candidates foruseful antagonists; or such antibodies may be used, e.g., in animmunoassay to monitor the level of LCF polypeptide produced by amammal, e.g., a human. Antibodies which antagonize LCF/CD4 receptorbinding or LCF mediated CD4 receptor function are considered to beuseful antagonists in the invention.

Identification of LCF in Bronchial Alveolar Lavage Fluid (BAL) ofAntigen Challenged Asthmatics

Below we describe the identification of two lymphocyte chemoattractantspresent in the BAL of asthmatics by 6 hrs following antigen challenge.One chemoattractant (LCF), a CD8⁺ cell product, acts exclusively on CD4⁺cells, while the second chemoattractant (MIP1α), a monocyte product,appears to act on both CD4⁺ and CD8⁺ cells. An important finding ofthese studies is that the chemical stimuli which result in T cellaccumulation in the lung in asthma are products of an inflammatorycascade which begins very early following antigen stimulation.Furthermore, we demonstrate that the majority of the lymphocytechemoattractant activity found in BAL fluid following antigen challengeis attributable to LCF. This example is intended to illustrate, notlimit, the invention.

Material and Methods

Subjects

Nine normal subjects (Table 1) and seven mild asthmatics (Table 2) wererecruited for the study in Southampton General Hospital. At the time ofenrollment all the asthmatic subjects had stable pulmonary function witha forced expiratory volume in one second (FEV₁) greater than 70% of thatpredicted for their age and height (Table 2). None of the asthmaticswere treated with inhaled or oral corticosteroids, sodium cromoglycateor theophylline for at least 6 weeks prior to their participation inthis study. They had hyperreactive airways to inhaled methacholine witha geometric mean provocative concentration of agonist required to reduceFEV₁ from baseline by 20% (PC₂₀) of 1.20 mg/ml (range 0.02-3.25 mg/ml).The asthmatic subjects were all atopic as defined by a >3mm skin whealresponse to one or more of 5 common allergens (Dermatophagoidespteronyssinus, mixed grass pollen, dog, feathers and cat dander(Hollister Stier). None of the asthmatics had experienced an upperrespiratory tract infection within six weeks of investigations.

Screening

Subjects attended the laboratory 4 days before the first bronchoscopywhen allergen skin prick testing, baseline spirometry and methacholinereactivity testing were performed. The technique used for bronchialchallenge was adapted from the 5 breath procedure of Chai et al. (J.Allergy Clin. Immunol. 56:323-327, 1975) using an Inspiron nebulizer (CRBard, Sunderland, U.K.). After recording baseline FEV₁, subjects inhaled5 breaths of 0.9% sodium chloride (saline) from functional capacity tototal lung capacity from the nebuliser via a mouthpiece. Measurements ofFEV₁ were made at 1 and 3 mins and, provided this value did not fallby >10% of baseline, the methacholine provocation was undertaken.Subjects inhaled sequential (doubled) concentrations (0.02-32 mg/mlsaline) of methacholine (Sigma Chemical Co.), with FEV₁ measurementsmade 1-3 mins after each inhalation. The stepwise methacholineinhalations continued until the FEV₁ had fallen by at least 20% of thepost-saline value. The concentration of methacholine was plotted againstthe percentage fall in FEV₁ from post-saline baseline, and thatconcentration causing a 20% fall in FEV₁ (PC₂₀) was derived by linearinterpolation of the last two data points.

The allergen used for local bronchial challenge (mixed grass pollen orD. pteronyssinus) was that which produced the largest wheal response onskin prick testing. In each subject a skin wheal dose response serieswas then undertaken using 10-fold dilutions of allergen and theconcentration chosen for the segmental bronchial challenge was one tenthof the dilution producing a 3 mm wheal response.

Bronchoscopy and local challenge

Study Design. Volunteers taking part in this study were divided into twogroups. A) Normal controls had a single bronchoscopy and BAL and, B)asthmatics had two bronchoscopies 6 hrs apart. Fiberoptic bronchoscopywas undertaken on subjects with FEV₁ greater than 70% of predicted, andthe platelet and clotting studies were within normal limits. Fiberopticbronchoscopy was undertaken using a standardized protocol. Subjectsreceived intravenous atropine (0.6 mg and midazolam 3-8 mg) prior tobronchoscopy. Oxygen (100%) was administered via nasal prongs throughoutthe procedure and oxygen saturation was monitored with a digitaloximeter (Minolta, Middlesex, U.K.). Fiberoptic bronchoscopy wasperformed with Olympus IT-20 bronchoscope (Olympus Optical Co., Tokyo,Japan). Care was taken to ensure that the larynx and upper airways wereadequately anesthetised using lignocaine spray (4%). The bronchoscopewas passed through the nares and up to 12 mls of lignocaine (1%) wasintroduced through the bronchoscope into the larynx and lower airways.Immediately after this procedure the bronchoscope was wedged into theanterior division of the right upper lobe (RUL) to undertake shamchallenge with 20 ml of sterile saline solution prewarmed to 37° C. Theinstrument was then passed into the medial subdivision of the rightmiddle lobe (RML) and 20 ml of prewarmed allergen solution wasinstilled. Five minutes after the introduction of the two solutions, theappearance of the airways was observed and photographed to record airwaynarrowing. Six hours later a second bronchoscopy was performed with thesame premedication and oxygenation. BAL was preformed with 6×20 mlaliquots of prewarmed 0.9% saline solution in both the allergen andsaline challenged bronchial segments. Returned fluid was aspiratedthrough the suction channel. Pulmonary function tests (FEV₁) wereperformed 3 hrs after the first bronchoscopy and 3 and 24 hrs after thesecond.

Lavage fluid processing

The recovered BAL fluid was centrifuged at 600 g for 15 min at 4° C.,the cells separated and the supernatant stored at -70° C. Lavage fluidswere concentrated a hundred fold by lyophilization following extensivedialysis, against ddH₂ O, using Spectapor membranes with a M.W.exclusion point of 3kDa.

Lymphocyte chemotaxis

Cell migration was performed according to standard methods (Cruikshanket al., J. Immunol. 138:3817-3825, 1987). Migration was assessed using amodification of the Boyden chamber assay using a microchemotaxis chamber(Neuroprobe, Cabin John, M.D.). Normal human T lymphocytes were isolatedusing hypaque-ficoll separation of peripheral blood mononuclear cellsfollowed by nylon wool adherence, resulting in >97% CD3⁺ cells by FACSanalysis, and then cultured overnight in RPMI 1640 containing 10% bovinefetal serum. The T cells (10×10⁶ /ml in RPMI 1640) were loaded into theupper well of the chamber, with 30 μl of the BAL fluids placed in thebottom chamber. The two wells were separated by a nitrocellulose filterpaper with a pore size of 8 μm. The chamber was incubated at 37° C. for3 hr, after which the filter was stained and migration was assessed bycounting the number of cells that had migrated beyond a certain depthinto the filter (50 μm). For most experiments between 15-20 cells/hpfwere counted in the control wells. In inhibition experiments thechemoattractant BAL was mixed with anti-cytokine antibodies (sufficientto neutralize bioactivity of 50 ng/ml of specific protein) for 30 min at37° C. prior to loading the chemotaxis chamber. All migration isexpressed as percentage values of cell migration in control buffer.

Antibodies

A rabbit polyclonal anti-rLCF antibody generated against a rLCF-KLHconjugate purified by protein A sepharose and rLCF affinitychromatography (described herein) was used for ELISAs, western analysisand lymphocyte migration inhibition studies according to standardmethods known in the art. Neutralizing anti-MIP1α, RANTES (R&D,Minneapolis, Minn.) and IL-8 (Endogen, Boston, Mass.) antibodies wereused according to manufactures specifications. These antibodies wereused at concentrations (anti-MIP1α at 20 ug/ml, anti-RANTES at 100ug/ml, and anti-IL-8 at 10 ug/ml) sufficient to neutralize lymphocytemigration induced by 50 ng/ml of the respective cytokines. There was nodetectable cross-neutralization between any of these antibodies for anyother cytokine tested.

ELISAs

ELISAS for LCF were performed using the antibody described above asfollows. Recombinant LCF and BAL samples were dissolved in PBS to theappropriate concentrations. Serial dilutions of rLCF were used for thestandard curve to which the unknowns were compared. 100 μl ofconcentrated samples were incubated in duplicate in a 96 well microtitreplate (Nunc) at 37° C. for 1 hr. All subsequent steps were conducted atroom temperature. The antigen was removed by washing four times with aPBS-Tween 20 solution. Non-specific binding was reduced by blocking with100 μl of 1% BSA for 1 hr. Following washing, 100 μl of a rabbitanti-LCF polyclonal antibody (10 μg/ml) diluted in PBS +0.05% Tween 20was added to each well. The presence of a LCF-anti-LCF complex wasdetected by the addition of biotinylated goat anti-rabbit IgG (Sigma)diluted 1:500 in PBS, incubated for 1 hr. After washing with PBS,ExtrAvidin peroxidase (Sigma) diluted 1:250 was incubated in each wellfor 30 mins, the plate was washed and 100 ul of freshly preparedsubstrate was added to the wells. The substrate consists of 0.2 mg/ml2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulphuric acid) in 0.05Mcitrate-phosphate buffer, pH 5.3, and 0.015% hydrogen peroxide. Thesubstrate was incubated in the dark for up to 30 min. The results of theELISA were read at 405 nm with a microplate reader. Using the Softmaxprogram the standard curve of known LCF was established and used todetermine the concentrations of LCF in BAL samples.

Quantitation for IL-3, IL-5, MIP1α, RANTES and GM-CSF levels were alsoassessed in the BAL samples using ELISAs. Commercial ELISA kits andreagents available from Genzyme (Cambridge, Mass.) for IL-3 and IL-5,Biosource International (Camarilo, Calif.) for GM-CSF, and R & D Systems(Minneapolis, Minn.) for MIP1α (Endogen, Boston, Mass.) were utilized.All commercial reagents were used according to manufacturersspecifications. For all cytokines, control species-specific-antibodieswere used to establish background levels. To more accurately quantitatecytokine concentrations, background levels were subtracted from samplemeasurements.

Results

Lymphocyte Chemotactic Activity From BAL Of Normal Individuals

We first determined whether lymphocyte chemoattractant activity could beidentified in the segmental BAL of normal individuals. The demographicsof the normal subjects are listed in Table 1.

                  TABLE 1    ______________________________________    Demagraphics at Normal Subjects    Subject  Age    Sex      FEV.sub.1.0 (%).sup.1                                     PC20 (mg/ml).sup.2    ______________________________________    N1       29     M        117.9   >32    N2       25     M        114.5   >32    N3       32     M        134.2   >32    N4       28     M        106.7   >32    N5       60     F        113.5   >32    N6       35     M        114.0   >32    N7       22     F        118.0   >32    N8       20     M        104.0   >32    N9       27     F        118.5   >32    ______________________________________     .sup.1 FEV.sub.1 % of predicted based on forced expiratory volume in the     1.sup.st second.     .sup.2 Methacholine concentration (mg/ml) required to produce 20% fall in     FEV.sub.1.

They all were non-atopic and had PC20's to methacholine of greater than32 mg/ml. We assayed the chemoattractant activity of unconcentrated andone hundred fold concentrated BAL to human peripheral T cells (FIG. 10).Samples were concentrated by first dialyzing against double distilledwater followed by lyophilization to effect a 100 fold concentration.Chemotaxis analyses of each undiluted sample did not demonstrate anyincrease in cell motility as compared with migration in the presence ofchemotaxis assay buffer alone. In fact, all of the samples wereinhibitory, with significant inhibition of migration (<80% of controlmigration, p<0.05) detected in 4 of 9 samples. The inhibitory effect didnot appear to mask the presence of T cell specific lymphocytechemoattractant activities of IL-8, RANTES, LCF or MIP1α, in any of theconcentrated BAL samples by ELISA (sensitivity >10 pg/ml and 40 pg/mlrespectively). Higher concentrations of normal BAL fluid were moreinhibitory; while greater dilutions were less inhibitory, eventuallyreaching (buffer) control cell migration at a dilution of 1:1,000. Fornone of the dilutions did BAL fluid from normals induce enhancedmigration. In addition there was no detectable lymphocytechemoattractant activity, for any dilution tested, in BAL six hoursfollowing saline challenge of 3 normals who volunteered for dualbronchoscopy studies. These dual BAL samples did demonstrate migrationinhibitory activity in a similar fashion as seen with BAL samplesobtained from a single lavage from normals.

Lymphocyte Chemoattractant Activity In BAL Fluid Of Asthmatics 6 hrFollowing Antigen Challenge

Table 2 describes the demographics of the asthmatic subjects.

                  TABLE 2    ______________________________________    Demographics and Characteristics of Asthmatics Subjects    Subject          Age     Sex    FEV.sub.1.sup.1                                PC20.sup.2                                      Allergen                                              (mg/ml)    ______________________________________    L1    27      M      84.1   3.45  grasses 10.sup.-4    L2    42      F      105.5  3.48  Derm. Ptery                                              10.sup.-5    L3    30      M      99.3   2.99  grasses 10.sup.-5    L4    33      F      104.0  1.47  Derm. Ptery                                              10.sup.-5    L5    25      M      76.2   2.28  Derm. Ptery                                              10.sup.-5    L6    37      F      88.9   2.96  grasses 10.sup.-5    L7    28      M      90.0   1.70  grasses 10.sup.-5    ______________________________________     .sup.1 FEV.sub.1 % of predicted based on forced expiratory volume in the     1.sup.st second.     .sup.2 Methacholine concentration (mg/ml) required to produce 20% fall in     FEV.sub.1.

FIG. 2 depicts the lymphocyte chemoattractant data from the BAL fluid ofasthmatics challenged with antigen as described above. While the BALfluid from normals demonstrated predominantly inhibitory activity,following saline challenge of asthmatics (FIG. 11) the subsequentconcentrated saline BAL were less inhibitory and, in some samples,chemoattractant activity was detected. Specifically, in individuals L1,L3, L4 and L6 the BAL following saline challenge reduced migration tobelow chemotaxis control, appearing like the BAL of normal individuals.The BAL fluid of the saline challenged lobes of individuals, L2 and L5,and L7 had some baseline lymphocyte chemoattractant activity; L7's BALfluid contained significant T cell chemoattractant activity. However, wedid detect an increase in lymphocyte chemoattractant activity in theantigen challenged, unconcentrated, subsegmental BAL from six of theseven asthmatics (FIG. 11) compared to their saline challenged lobes(p<0.05 for each except L7). Four of the seven antigen samples (L1, L2,L4, and L5) demonstrated significant increases in migration compared tocontrol migration buffer. BAL samples L3b and L6b did induce significantincreased migratory responses compared to their saline control (p<0.05)but compared to chemotaxis buffer, the levels did not reach statisticalsignificance (p>0.05). None of the saline or antigen challenge solutionshad any intrinsic lymphocyte chemoattractant activity.

As shown in Table 3, there were no detectable changes in celldifferentials following antigen challenge. On average the recovered cellpopulation in the BAL fluid remained consistent pre- and post-antigenchallenge with cell differentials of 65-70% macrophages, 13-20%lymphocytes, 17-21% neutrophils, and 4-10% eosinophils. This findingsuggests that the cytokines present in the BAL fluid at the 6 hr timepoint were produced by either pre-existing or newly recruited cells atthe site of antigen challenge. This also indicates that chemoattractantsdetected at this early time point play a role in recruitment ofresponding cells.

                  TABLE 3    ______________________________________    Differential Cell Counts 6 hrs Post Saline or Allergen Challenge.sup.1    Sub- Macro-  Lympho-  Neutro-      Cells Total    jects         phage   cyte     phil  Eosinophil                                       Counted                                             Cells/ml    ______________________________________    L1a.sup.2         522     108      139   31     800   9.6 × 10.sup.4    L1b.sup.3         539     126      93    45     803   9.6 × 10.sup.4    L2a  557     195      48    5      805   6.8 × 10.sup.4    L2b  271     85       403   46     805   8.2 × 10.sup.4    L3a  515     237      47    2      801   6.8 × 10.sup.4    L3b  453     162      195   4      814   4.0 × 10.sup.4    L4a  457     284      59    5      805   13.9 × 10.sup.4    L4b  384     289      118   13     804   25.9 × 10.sup.4    L5a  411     226      167   1      805   8.8 × 10.sup.4    L5b  552     118      84    52     806   7.9 × 10.sup.4    L6a  750     25       22    7      804   2.0 × 10.sup.4    L6b  640     23       137   10     810   3.3 × 10.sup.4    L7a  291     236      270   22     819   11.3 × 10.sup.4    L7b  460     173      105   65     803   8.6 × 10.sup.4    ______________________________________     .sup.1 Cell counts were performed on recovered BAL fluid from an     instillation volume of 120 ml. Following centrifugation, cell pellets wer     cytospin centrifuged and stained with a Wright'sGiemsa`s stain.     .sup.2 Cells obtained 6 hrs following saline challenge are designated by     an "a".     .sup.3 Cells obtained 6 hrs following antigen challenge are designated by     a "b".

Characterization Of The Lymphocyte Chemoattractant Activity

Initial characterization of the chemoattractant activity from the BAL ofantigen challenged lobes was conducted using neutralizing antibodies toknown lymphocyte chemoattractants. Neutralizing antibodies to LCF, IL-8,MIP1α, and RANTES were used in this study. Co-incubation of the BALsamples with 1 μg/ml anti-LCF polyclonal antibody (sufficient toneutralize 5 ng/ml of LCF bioactivity) for 30 min prior to thechemotaxis assay, reduced the chemoattractant activity for each of thesamples (FIG. 12). However, anti-LCF antibody did not completely inhibitthe migratory response to any of the BAL samples, as compared with thesaline control values, indicating that other chemoattractants were alsopresent. Co-incubation with neutralizing antibodies for IL-8 or RANTESbioactivity had no effect on the lymphocyte chemoattractant activity,while antibodies to MIP1α did have an inhibitory effect. (FIG. 12).Samples L2b and L4b demonstrated the greatest percent blocking byanti-MIP1α alone. A combination of anti-LCF and anti-MIP1α inhibited90-95% of all induced migration of each of the BAL's following antigenchallenge (FIG. 13). The addition of the other antibodies to eitheranti-LCF or anti-MIP1α did not further reduce the migratory response.Interestingly, individual L7 had demonstrated significantchemoattractant activity following saline challenge. When this samplewas co-incubated with anti-LCF or anti-MIP1α antibodies, the migratoryresponse was partially reduced (FIG. 12). A combination of anti-LCF andanti-MIP1α did not completely eliminate all induced migration (FIG. 13)indicating that other chemoattractants are present. Overall, the majorlymphocyte chemoattractant activity was LCF in the antigen challengedBAL. In total, approximately 90-95% of all the chemoattractant activitywas attributed to a combination of LCF and MIP1α.

Detection Of LCF And MIP1α Protein By ELISA.

We next determined whether these same samples had detectable levels ofLCF and MIP1α protein. Table 4 shows the results of these ELISAs of theBAL from antigen and saline challenged lobes expressed as pg/ml of totalBAL volume. The LCF concentrations were determined from concentrated BALcorrected to starting volume.

                  TABLE 4    ______________________________________    ELISA-quantitated LCF and MIP1α Concentration in BAL.sup.1 in    asthmatic    subjects           LCF (pg/ml)       MIP1α (pg/ml)    Subjects Saline  Antigen     Saline                                       Antigen    ______________________________________    L1       <10     1003        <40   <40    L2       <10     7042        <40   503    L3       <10     <10         <40   75    L4       <10     11035       <40   720    L5       <10     9052        <40   95    L6       <10     958         <40   88    L7       208     64          55    73    ______________________________________     .sup.1 BAL samples were concentrated from 50 ml to 0.5 ml by spin     centrifugation. The ELISA for LCF was conducted using a protein A purifie     rabbit antirLCF antibody, and detected by a biotinconjugated goat     antirabbit polyclonal antibody. The sensitivity for the assay ranged from     10 pg/ml to 20 ng/ml. The MIP1α ELISA, available from R and D     Systems, was conducted using the manufacturers specifications. The     sensitivity of the assay ranged from 40 pg/ml to 2 ng/ml. Each sample was     run in triplicate and the data represents the average of the three values

One hundred microliters of each BAL sample was assayed concentrated 100fold from the original volume of lavage fluid. The data are expressed aspg/ml of BAL sample corrected to the orginal BAL volume. Allergenchallenged asthmatic BAL L2b, L4b and L5b had values close to 1 ng/ml.For L2b this was an LCF protein concentration of 47 ng/ug of totalprotein (total protein assessed by Bradford Protein Analysis), for L5b,13.5 ng/ug total BAL protein and 1.5 ng/ml for sample L4b which was 89ng/ug unconcentrated BAL protein. We did not detect any LCF fromconcentrated BAL of saline challenged lobes, and subjects 3, and 6 hadundetectable LCF by ELISA in their antigen challenged lobes. This isconsistent with the relative amount of LCF-induced bioactivity observedfor these samples and the greater sensitivity of the chemotaxis assay ascompared with the ELISA.

Detectable levels of RANTES were not observed at the 6hr time pointfollowing either saline or antigen challenge in the BAL fluid. Thesedata combined with the lack of neutralizing effects by the anti-RANTESantibodies indicates that RANTES were not present at the 4-6 hr timepoint.

The quantitation of MIP1α protein by ELISA is also shown in Table 3. Thetwo BAL samples which demonstrated the largest percent blocking byanti-MIP1α antibodies, L2b and L4b, exhibited MIP1α protein in the rangeof 600 pg/ml (corrected to original BAL volume) (Table 3). Several othersamples, L3b, L5b, and L7b, had trace amounts of detectable MIP1αprotein. For both chemoattractants the samples which displayed thegreatest chemoattractant activity also demonstrated the highestdetectable levels of protein. At this six hour time point followingantigen challenge there were no measureable levels of cytokines IL-3,IL-5 or GM-CSF by ELISA, with a lower sensitivity limit of 40 pg/ml, inaliquots of the same BAL samples. Detectable levels of IL-8 protein werepresent in all antigen challenge and most saline challenged lobes.

Stimulation of Cell Division Using LCF and A Growth Factor

We have discovered that recombinant LCF induces the expression of cellreceptors, e.g., IL-2R, which subsequently render a cell-bearing thereceptor, e.g., a T cell, competent to respond to its cognate growthfactor, e.g., IL-2. In one working example, human T cells werestimulated with recombinant LCF (a concentration range of 10⁻⁵ M to10⁻¹⁰ M was used with similar results, data for 10⁻⁸ M is shown) for 24h at which time rIL-2 (2U/ml) or anti-CD3 (OKT3, 50 ng/ml) were added tothe cell cultures. Four days after the addition of either rIL-2 or OKT3antibody cell proliferation was assayed by ³ H thymidine uptake.Averaging the results of all three experiments shown in Table 5, showingthe effects of recombinant LCF on anti--CD3 and rIL-2 induced thymidineincorporation, recombinant LCF preincubation resulted in enhanced IL-2responsiveness. Human T cells do not increase the incorporation of ³ Hthymidine following incubation with recombinant LCF alone at either 24or 48 h, but following preincubation with recombinant LCF, rIL-2stimulated T cells increase their incorporation of ³ H thymidine from1,079 cpm to 13,818 cpm. However, in the recombinant LCF treated cellcultures the proliferative response to anti-CD3 antibody was reducedapproximately 50% from 21,257 cpm for anti-CD3 stimulation alone to 12,047 cpm in cell stimulated with recombinant LCF.

Thus, in the example given human T cells were incubated with recombinantLCF for 24 hours prior to stimulation with the T cell growth factorinterleukin 2. Prior incubation of T cells with recombinant LCF resultedin a 5 fold increase in incorporation of ³ H-thymidine (DNA synthesis)at 72 hours compared to either recombinant LCF or rIL-2 alone. Thissynergy was specific for IL-2 as prior incubation of T cells withrecombinant LCF decreased subsequent ³ H-thymidine incorporation inresponse to T cell antigens (see anti-CD₃ responses).

                  TABLE 5    ______________________________________    Stimulus   Expt. 1    Expt. 2     Expt. 3    ______________________________________    Control     983 ± 145                          1074 ± 326                                       946 ± 197    LCF(10.sup.-8 M)               1203 ± 284                          1054 ± 212                                       982 ± 301    Anti-CD3(50 ng/ml)               22485 ± 1077                          20496 ± 998                                      20792 ± 1048    rIL-2(1U/ml)               2381 ± 185                          2594 ± 464                                       2508 ± 4071    LCF + anti-CD3*               12497 ± 1038                          11739 ± 335                                      11905 ± 1127    LCF + rIL-2*               12664 ± 2802                          15037 ± 1088                                      13753 ± 2068    ______________________________________     *Cultures were stimulated with LCF for 24 hr prior to the addition of     either antiCD3 antibody or rIL2. Cultures were conducted for a total of 5     days.

Next, we evaluated the effect of media, rIL-2, rLCF, and rLCF on humannylon wool non-adherent T cells (NWNAT) (Julius et al., Eur. J. Immunol.3:645, 1973) proliferation under long term culture conditions. Theresults of eight individual experiments (performed in duplicate) areshown in FIG. 14. Cells were plated at 3×10⁶ /ml in one ml cultures. Inthe rLCF culture and the rIL-2 culture, the cytokines were added everytwo days. The dosing schedule for rLCF/rIL2 was rLCF (10⁻¹⁰ M) eachMonday, followed every Wednesday and Friday by rIL-2 (10 U/ml). Cellcounts were performed once every week.

The mean total of numbers of cells present in the one ml cultures aredepicted on the ordinate of FIG. 14. In the cultures containing onlymedia and cultures treated with rIL-2 alone, cell numbers declined byhalf. Treatment with rLCF preserved cell numbers, while treatment withrLCF and rIL-2 increased cell numbers to over twice the original platingdensity, resulting in nearly four times greater mean cell numbers foundin cultures treated with rIL-2 or untreated cells at six weeks.

We next examined the effect of the above treatments using long term HIV+PBMCs. The growth kinetics of long term PMBC cultures obtained from fiveHIV+ individuals are shown in FIG. 15. Using the culture conditionsdescribed above, we examined the effect of rLCF, rIL-2, rLCF followed byaddition of rIL-2, or media alone on the viability and proliferation ofHIV+ PBMCs. Cultures were plated at 3×10⁶ per ml and maintained in oneml cultures with the addition of cytokines as outline above. The resultsshown in FIG. 15 demonstrate that PBMCs of HIV+ patients in unstimulatedculture, IL-2 treated, or rLCF treated cultures undergo rapid early celldepletion and by 6-7 weeks more than 90% of the cells have lysed.Compared to untreated cells, IL-2 at 10 u/ml did not lead to aproliferative response. In addition, rLCF did not increase cell survivalnor increase the rate of cell lysis when compared to control or IL-2treatments. The results also show that cells cultured with rLCF(10⁻¹⁰ M)in combination with IL-2(10 U/ml) not only survive, but proliferate forat least 9 weeks. The difference in cell survival between combined rLCFand rIL-2 and control, using paired t-test analysis, is statisticallysignificant at p=0.02. All of these surviving and proliferating cellswere CD4+.

These data were then evaluated in the context of the patient's CD4 countshown in FIG. 16. The data shown in FIG. 16 demonstrate that theproliferative response of HIV+ PBMCs due to rLCF and IL-2 treatment isgreater in those patients having CD4 counts over 50. The results alsoappear to suggest that the cultures have a lower chance of survival whenthe CD4 count is low. (The two curves where the cultures did not survive(Data for curves 5 and 6) belong to the same patient, and that the CD4count had not changed during the month interval between theexperiments.) As shown in FIG. 16, all the patients have CD4 counts ofless than or equal to 500. Of these, 3/4 of the patients that had thegreater proliferative response had a CD4 count of ≧290. The CD4/CD8ratio for all the individuals ranged between 0.1 and 0.4. Furthermore,three patients with the highest proliferative response, also had thehighest ratios, 0.3 and 0.4.

The loss of cells beyond week 9 suggests that HIV infection has anadverse effect on these cultures. To further investigate this decline incells, we performed p24 ELISA measurements on the cultures according tomethods known in the art. As shown in FIG. 17, there is a four-foldincrease in the p24 measurements, indicating that while cells areactivated and induced to proliferate, the virus is also proliferating.Accordingly, it is possible that by week 9 viral replication leads tocell lysis. Given this result, an anti-retroviral agent, e.g., AZT orddI, should be included in the culture to reduce viral load.

These results demonstrate that there is both survival and proliferationof HIV+ PBMCs cultures when treated with rLCF and rIL-2. Based on ourdiscovery (described above) that rLCF preserves CD4+ T cell numbers andin combination with rIL-2 increases CD+4 T cells, therapies, in vivo orex vivo or both, are made available for reconstituting the immunesystem, e.g., by preserving, increasing, and/or expanding the number ofCD4+ cells, as described herein.

In one working example, evaluation of whether ex vivo and in vivotreatment using rLCF in combination with a growth factor, e.g., rIL-2,confers protection against the development of an immune disorder, e.g.,AIDS, generally involves using standard animal models (e.g., Hu-PBL-SCIDor the Hu-PBL-SCID-HIV mouse models). For evaluation of in vivo therapy,an appropriate animal is treated with rLCF and a growth factor, e.g.,rIL-2, and if desired, an antiretroviral agent such as AZT or ddI,according to any standard method, and a reduced incidence of thedevelopment of an immune disorder, e.g., AIDS, compared to untreatedcontrol animals, is detected as an indication of protection. In general,the scheduling for administering rLCF and a growth factor follows thescheduling of adding rLCF and rIL-2 to cell cultures described above.This protocol is then repeated, as necessary. Alternatively, rLCF and agrowth factor are administered simultaneously. Recombinant compositionsand antiviral agents are administered according to methods known in theart, e.g., by intravenous or subcutaneous injection, inpharmaceutically-acceptable formulations. Optimization of the dosingparameters and scheduling the administration of the LCF and the growthfactor is carried out according to methods known in the art.

Alternatively, for evaluation of ex vivo therapy, PMBCs are collectedfrom an appropriate animal on the day the blood is drawn according tostandard techniques. For example, blood samples are diluted in equalvolume phosphate-buffered saline and layered over a ficoll/hypaquedensity gradient. After centrifugation, the PMBC suspension is aspiratedfrom the interface. Collected PMBCs are washed in PBS and viability andcell numbers are determined by microscopy according to standardtechniques, e.g., using trypan blue and Turk's crystal violet stains.PMBC suspensions are brought to volume in PBS to be used forreconstitution of an appropriate animal model, e.g., SCID, and treatedwith rLCF and a growth factor, and, if desired, an antiviral agent, asdescribed above. Treatment of the cell cultures is continued, e.g., for8-12 weeks, after which time the cells are reintroduced to the donoranimal. If necessary the process is repeated. Alternatively, the rLCFand growth factor are administered simultaneously. Animals receiving exvivo treatments having a reduced incidence of the development of animmune disorder, e.g., AIDS, compared to untreated control animals, arean indication of protection. Optimization of the dosing parameters andscheduling the administration of the LCF and the growth factor iscarried out according to methods known in the art.

LCF Anatagonists As Anti-Cancer Agents

Anti-cancer agents of the invention, e.g., LCF antagonists (as describedabove, for example, a fragment or analog of LCF, or an anti-LCFantibody) are useful for inhibiting a neoplasm, e.g., CD4+ leukemias orlymphomas, or any malignant cell bearing CD4+ receptors. Those skilledin the art will understand that any number of methods, both in vitro andin vivo, are used to determine the efficacy of anti-cancer agents usefulin the methods of the invention. For example, the reduction ofneoplasmic growth can be monitored in a mouse or rat growing a cancerfollowing the administration of the test compound. In a working example,a neoplasmic cell line growing in culture (e.g., those cell linesdescribed herein) is released from monolayer by trypsinization, dilutedinto single-cell suspension and then solidified by centrifugation into apellet which is subsequently exposed to 15 μl fibrinogen (50 mg/ml) and10 μl thrombin (50 units/ml) for 30 minutes at 37° C. Fibrin clotscontaining tumor are then cut into pieces approximately 1.5 mm indiameter. Each piece of tumor is subsequently implanted under the kidneycapsule of a mouse according to standard methods. Generally,administration of the test molecule is initiated prior to neoplasmicimplantation and/or after neoplasmic implantation. Control animalsreceive a placebo, e.g., serum albumin or diluent, similarlyadministered as for the LCF inhibitor or related molecules. The effectof the test molecule on neoplasmic growth is monitored according to anystandard method. A molecule shown experimentally to halt or reduce orinhibit the growth of such an implanted neoplasm is considered useful inthe invention.

Evaluation of whether a test compound confers protection against thedevelopment of a neoplasm (e.g., CD4+ leukemias or lymphomas) alsoinvolves using an animal known to develop a neoplasm (e.g., thetransgenic mouse described in U.S. Pat. No. 4,736,866). An appropriateanimal is treated with the test compound according to standard methods,and a reduced incidence of neoplasm development, compared to untreatedcontrol animals, is detected as an indication of protection.

Alternatively, the evaluation of whether a test compound confersprotection against the development of a neoplasm is evaluated in vitroaccording to any standard method known in the art. Cell lines useful forexamining the in vitro effects of a LCF inhibitor include, withoutlimitation, SUP-TI (ATCC CRL 1942); J45.01 (ATCC CRL 1990); J-111(ATCCCRL 8129); J-A1886 (ATCC CRL 8130); 8E5 (ATCC CRL 8993); C5/MJ (ATCC CRL8293; D10.G4.1 (ATCC TIB 224); HVS-SILVA 40 (ATCC CRL 1773); CTLL-2(ATCC TIB 214); HuT 102 (ATCC TIB 162); Mo (ATCC CRL 8066); Mo-B (ATCCCCL 245); HUT 78 (ATCC TIB 161); and THP 1 (ATCC TIB 202). A moleculeshown experimentally to halt or reduce or inhibit the growth of suchcell lines is considered useful in the invention.

As is discussed below, we have discovered that an LCF inhibitor has beenfound to be a potent inhibitor of neoplasmic THP1 growth in vitro. Theexperimental example described below demonstrates the efficacy ofanti-LCF antibodies as an anti-cancer agent. This example is provided toillustrate, not limit, the invention.

Our finding that LCF demonstrates a CD4 dependent transition from G_(o)to G₁ (marked by the induction of IL-2R and HLA-DR) prompted us topursue the possibility that this response might be TcR/CD3 independent.The chemotactic response of TcR(-) monocytes and eosinophils furthersuggested that the presence of CD3 is not an absolute requirement forCD4 signaling. In order to pursue this hypothesis we utilized aCD4+CD8+CD3- T cell line Sup-T1, which we found to exhibit a motileresponse to LCF and Leu3a antibodies. In addition, we found that the LCFinduced response is inhibited by both anti-LCF antibodies and rsCD4.

To determine if Sup-T1 was a suitable cell line to dissect thedifferences between motility and growth signals we investigated theeffects of LCF and LCF inhibition on SupT1 cell growth. This experimentwas carried out as follows. Sup T1 cells were cultured in the presenceor absence of LCF for 24 hrs according to standard methods, after whichtime the cells were loaded with the metachromatic dye, acridine orange,for determination of DNA and RNA content. The results showed that thereis a marked cell cycle change with many cells progressing into S, G₂ andM. This finding is in contrast to normal T cells which progress only asfar as G₁ following stimulation with LCF. In addition, we also examinedthe ability of Sup-T1 to take up ³ H thymidine. For these experiments,5×10⁵ cells per well were cultured for 24 hrs with or without theaddition of either rLCF or monoclonal anti-LCF antibody. The wells werepulsed for 8 hrs with 1 μCi ³ H-thymidine and quantitated byscintillation counting. Data obtained from these experiments are shownin Table 6 (the average of three sets of experiments each performed intriplicate). In addition, Northern analysis failed to detect any messagefor either IL-2, IL-4 or IL-2R. Accordingly, the growth of Sup T1 isIL-2 and IL-4 independent, but it can be altered by LCF interaction withCD4.

                  TABLE 6    ______________________________________    Effects of rLCF and Anti-LCF Antibody on Supt1 and    THP1 Growth                       +anti-LCF    Cells Alone        (10 ug/ml)   +LCF (10.sup.-8 M)    ______________________________________    SUPT1 37,835 +/- 968                       39,804 +/- 1175                                    59,804 +/- 1175    THP1  21,847 +/- 1137                       10,592 +/- 1091                                    29,478 +/- 1284    ______________________________________

Experiments investigating the effects of LCF and anti-LCF antibodies ongrowth of the CD4+ receptor bearing cell line are shown in Table 6. Wefound that additional exogenous LCF increased ³ H thymidineincorporation. Moreover, THP1 cells cultured in the presence of anti-LCFantibody alone decreased the normal ³ H thymidine incorporation of thesecells by at least 50% (Table 6). This response is IL-2R/IL-2 independentas anti-IL-2 and anti-Tac have no effect on the LCF-CD4 related cellcycle changes.

As demonstrated above, anti-LCF antibodies are effective in inhibitingneoplasmic growth of CD4+ cells, e.g., THP1, and SUP-T1. Accordingly,compounds of the invention can be formulated according to known methodsto prepare pharmaceutically useful compositions as described herein.Treatment of human patients will be carried out using a therapeuticallyeffective amount of an anti-cancer agent of an LCF inhibitor in aphysiologically acceptable carrier. Suitable carriers and theirformulation are described for example in Remington's PharmaceuticalSciences by E. W. Martin. The amount of the anti-cancer agent to beadministered varies depending upon the manner of administration, the ageand body weight of the patient, and with the type of disease,extensiveness of the disease, and size of the patient suffering from thedisease. Generally amounts will be in the range of those used for otheragents used in the treatment of cancer, although in certain instanceslower amounts will be needed because of the increased specificity of thecompound. For example, an anti-LCF antibody is administeredsystemically, as described below, at a dosage that inhibits malignantcell proliferation, typically in the range of 0.1 ng-10 g/kg bodyweight.

Furthermore, the method of the invention can also employ combinationtherapy in which an LCF inhibitor is administered either simultaneouslyor sequentially with a chemotherapeutic agent. Typically, achemotherapeutic agent is administered according to standard methodsor,alternatively, in a dose which is lower than the standard dose whenthe chemotherapeutic agent is used by itself. Examples ofchemotherapeutic agents include, without limitation, mechlorethamine,cyclophosphamide, ifosfamide, L-sarcolysin, chlorambucil,hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine,semustine, streptozocin, dacarbazine, methotrexate, fluorouracil,cytarabine, mercaptopurine, thioguanine, pentostatin, vinblastine,vincristine, etoposide, teniposide, actinomycin D, daunomycin,doxorubicin, bleomycin, plicamycin, mitomycin, cisplatin, mitoxantrone,hydroxyurea, procarbozine, mitotane, aminoglutethimide, prednisone,hydroxyprogesterone, diethylstilbestrol, tamoxifen, flutamide, orleuprolide. Treatment is started generally with the diagnosis orsuspicion of a neoplasm and is generally repeated on a daily basis.Protection from the development of a neoplasm is also achieved byadministration of an LCF inhibitor on a daily basis. If desired, theefficacy of the treatment or protection regimens is assessed with themethods of monitoring or diagnosing patients for cancer. Furthermore,the anti-cancer compounds of the invention can also be used to treatmammals to destroy any unwanted cells bearing CD4+ receptors associatedwith a pathological condition.

LCF Kits

Kits for carrying out the above methods and using the above compositionsare also included in the invention. Such kits preferably include asubstantially pure antibody that specifically recognizes and binds a LCFpolypeptide, and may also include means for detecting and quantitatingantibody binding. Alternatively, the kit may include all or a fragmentof a LCF nucleic acid sequence useful for hybridization purposes, andmay also include means for detecting and quantitating LCF RNAhybridization.

Therapy

Particularly suitable therapeutics for the treatment of hyperresponsiveimmune responses and inflammatory diseases are the soluble antagonisticfragments described above formulated in an appropriate buffer such asphysiological saline. Furthermore, anti-LCF polypeptide (fragments oranalogs thereof) antibodies produced as described above may be used astherapeutics. Again, the antibodies would be administered in apharmaceutically-acceptable buffer (e.g., physiological saline). Ifappropriate, the antibody preparation may be combined with a suitableadjuvant. Similarly, the methods of the invention provide for theidentification of an organic compound useful to antagonize LC4:CD4interaction, once identified and isolated such a compound can then beformulated in an appropriate buffer and used as a therapeutic.

In addition, suitable therapeutics for the use of LCF or LCF agonists asimmunosuppressive agents or as therapeutics to stimulate the expansionof CD4+ receptor bearing cells (as described supra) are formulated in anappropriate buffer such as physiological saline. Again, theseformulations would be administered in a pharmaceutically-acceptablebuffer (e.g., physiological saline).

Ordinarily, the therapeutic composition will be administeredintravenously, at a dosage effective to stimulate activation of new CD4lymphocyte populations; to induce anergy (see table above) and inhibitrejection in transplants; and to attenuate a hyperresponsive immuneresponse and inflammation, e.g., asthma.

Alternatively, it may be convenient to administer the therapeuticorally, nasally, or topically, e.g. as a liquid or a spray as a primaryproduct or as a viral vector carrying LCF cDNA. Again, the dosages areas described above. However, the dosage of the compound for treating anyof the above-mentioned disorders varies depending upon the manner ofadministration, the age and the body weight of the subject, and thecondition of the subject to be treated, and ultimately will be decidedby the attending physician or veterinarian. Such amount of the activecompound as determined by the attending physician or veterinarian isreferred to herein as a "therapeutically effective amount." Thecompounds of the invention can be administered to a mammal, e.g., ahuman patient in a dosage of 0.5 μg/kg/day to 5 mg/kg/day.

Synergistic effect between recombinant LCF and growth factor (e.g.,IL-2) are induced by sequential administration of recombinant LCF (0.5μg/kg to 5 mg/kg followed in 24 hours by similar doses or rIL-2. Asdemonstrated above, rLCF and rIL-2 are effective in promoting thesurvival and proliferation of immune cells, e.g., HIV+ PBMCs.Accordingly, compounds of the invention can be formulated according toknown methods to prepare pharmaceutically useful compositions. Treatmentof human patients will be carried out using in vivo and/or ex vivoadminstration of a therapeutically effective amount of rLCF and IL-2.Suitable carriers and their formulation are described for example inRemington's Pharmaceutical Sciences by E. W. Martin. The amount of therLCF and IL-2 to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with thetype of disease, extensiveness of the disease, and size of the patientsuffering from the disease. Generally amounts will be in the range ofthose used for other agents used in the treatment of other immunedisease, e.g., AIDS. For example, for rLCF and rIL-2 is administeredsystemically, as at a dosage that promotes cell proliferation, typicallyin the range of 0.5 μg/kg/day to 5 mg/kg/day. Treatment is startedgenerally with the diagnosis or suspicion of an immune disorder and isgenerally repeated on a daily basis. Protection from the development ofan immune disorder, e.g., AIDS, is also achieved by administration ofrLCF on a daily basis, and if desired, in combination with rIL-2. Ifdesired, the efficacy of the treatment or protection regimens isassessed with standard methods of monitoring or diagnosing patients foran immune disorder. Reconstitution of the immune system, e.g., apatient's CD4+ cells, is useful in cellular immunotherapy forpreventing, suppressing, or inhibiting the failure of the immune system,e.g, as found during HIV infection. In general, such treatment is usefulfor treating or delaying additional immunological and clinicaldeterioration.

The methods of the invention may be used to reduce the disordersdescribed herein in any mammal, for example, humans, domestic pets, orlivestock. Where a non-human mammal is treated, the LCF polypeptide orthe antibody employed is preferably but not necessarily specific forthat species.

OTHER EMBODIMENTS

The invention includes any protein which is substantially homologous toLCF polypeptide (FIG. 2, SEQ ID NO: 1). LCF is expressed in human Tcells and exocrine pancreas. It is also expressed in the humanmonocytoid cell line THP-1. Also included are: allelic variations;natural mutants; induced mutants; proteins encoded by DNA thathybridizes under high or low (e.g., washing at 2×SSC at 40° C. with aprobe length of at least 40 nucleotides) stringency conditions to anucleic acid naturally occurring (for other definitions of high and lowstringency see Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1989); and polypeptides or proteins specifically boundby antisera to LCF polypeptide, especially by antisera to the activesite or binding domain of LCF polypeptide. The term also includeschimeric polypeptides that include LCF polypeptide.

In addition to substantially full-length polypeptides, the inventionalso includes biologically active fragments of the polypeptides. As usedherein, the term "fragment", as applied to a polypeptide, willordinarily be at least about residues, more typically at least about 40residues, preferably at least about 60 residues in length. Fragments ofLCF polypeptide can be generated by methods known to those skilled inthe art. The ability of a candidate fragment to exhibit a biologicalactivity of LCF polypeptide can be assessed by methods known to thoseskilled in the art as described herein. Also included are LCFpolypeptides containing residues that are not required for biologicalactivity of the peptide such as residues that are not required for thebiological activity of the polypeptide, or that result from alternativemRNA splicing or alternative protein processing events.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 130 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetProAspLeuAsnSerSerThrAspSerAlaAlaSerAlaSerAla    151015    AlaSerAspValSerValGluSerThrAlaGluAlaThrValCysThr    202530    ValThrLeuGluLysMetSerAlaGlyLeuGlyPheSerLeuGluGly    354045    GlyLysGlySerLeuHisGlyAspLysProLeuThrIleAsnArgIle    505560    PheLysGlyAlaAlaSerGluGlnSerGluThrValGlnProGlyAsp    65707580    GluIleLeuGlnLeuGlyGlyThrAlaMetGlnGlyLeuThrArgPhe    859095    GluAlaTrpAsnIleIleLysAlaLeuProAspGlyProValThrIle    100105110    ValIleArgArgLysSerLeuGlnSerLysGluThrThrAlaAlaGly    115120125    AspSer    130    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2150 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TTCCTCGAGAGCTGTCAACACAGGCTGAGGAATCTCAAGGCCCAGTGCTCAAGATGCCTA60    GCCAGCGAGCACGGAGCTTCCCCCTGACCAGGTCCCAGTCCTGTGAGACGAAGCTACTTG120    ACGAAAAGACCAGCAAACTCTATTCTATCACCAGCCAGTGTCATCGGCTGTCATGAAATC180    CTTGCTGTGCCTTCCATCTTCTATCTCCTGTGCCCAGACTCCCTGCATCCCCAAGGCAGG240    GGCATCTCCAACATCATCATCCAACGAAGACTCAGCTGCAAATGGTTCTGCTGAAACATC300    TGCCTTGGACACGGGGTTCTCGCTCAACCTTTCAGAGCTGAGAGAATATACAGAGGGTCT360    CACGGAAGCCAAGGAAGACGATGATGGGGACCACAGTTCCTTCAGTCTGGTCAGTCCGTT420    ATCTCCCTGCTGAGCTCAGAAGAATTAAAAAAACTCATCGAGGAGGTGAAGGTTCTGGAT480    GAAGCAACATTAAAGCAATTAGACGGCATCCATGTCACCATCTTACACAAGGAGGAAGGT540    CGTGGTCTTGGGTTCAGCTTGGCAGGAGGAGCAGATCTAGAAAACAAGGTGATTACGGTT600    CACAGAGTGTTTCCAAATGGGCTGGCCTCCCAGGAAGGGACTATTCAGAAGGGCAATGAG660    GTTCTTTCCATCAACGGCAAGTCTCTCAAGGGGACCACGCACCATGATGCCTTGGCCATC720    CTCCGCCAAGCTCGAGAGCCCAGGCAAGCTGTGATTGTCACAAGGAAGCTGACTCCAGAG780    CCATGCCCGACCTCAACTCCTCCACTGACTCTGCAGCCTCAGCCTCTGCAGCCAGTGATG840    TTTCTGTAGAATCTACAGCAGAGGCCACAGTCTGCACGGTGACACTGGAGAAGATGTCGG900    CAGGGCTGGGCTTCAGCCTGGAAGGAGGGAAGGGCTCCCTACACGGAGACAAGCCTCTCA960    CCATTAACAGGATTTTCAAAGGAGCAGCCTCAGAACAAAGTGAGACAGTCCAGCCTGGAG1020    ATGAAATCTTGCAGCTGGGTGGCACTGCCATGCAGGGCCTCACACGGTTGGAAGCCTGGA1080    ACATCATCAAGGCACTGCCTGATGGACCTGTCACGATTGTCATCAGGAGAAAAAGCCTCC1140    AGTCCAAGGAAACCACAGCTGCTGGAGACTCCTAGGCAGGACATGCTGAAGCCAAAGCCA1200    ATAACACACAGCTAACACACAGCTCCCATAACCGCTGATTCTCAGGGTCTCTGCTGCCGC1260    CCCACCCAGATGGGGGAAAGCACAGGTGGGCTTCCCAGTGGCTGCTGCCCAGGCCCAGAC1320    CTTCTAGGACGCCACCCAGCAAAAGGTTGTTCCTAAAATAAGGGCAGAGTCACACTGGGG1380    CAGCTGATACAAATTGCAGACTGTGTAAAAAGAGAGCTTAATGATAATATTGTGGTGCCA1440    CAAATAAAATGGATTTATTAGAATTCCATATGACATTCATGCCTGGCTTCGCAAAATGTT1500    TCAAGTACTGTAACTGTGTCATGATTCACCCCCAAACAGTGACATTTATTTTTCTCATGA1560    ATCTGCAATGTGGGCAGAGATTGGAATGGGCAGCTCATCTCTGTCCCACTTGGCATCAGC1620    TGGCGTCATGCAAAGTCATGCAAAGGCTGGGACCACCTGAGATCATTCACTCATACATCT1680    GGCCGTTGATGTTGGCTGGGAACTCACCTGGGGCTGCTGGCCTGAATGCTTATAGGTGGC1740    CTCTCCTTGTTGCCTGGGCTCCTCACAACATGGTGTCTGGATTCCCAGGATGAGCATCCC1800    AGGATCGCAAGAGCCACGTAGAAGCTGCATCTTGTTTATACCTTTGCCTTGGAAGTTGCA1860    TGGCATCACCTCCACCATACTCCATCAGTTAGAGCTGACACAAACCTGCCTGGGTTTAAG1920    GGGAGAGGAAATATTGCTGGGGTCATTTATGAAAAATACAGTTTGTCACATGAAACATTT1980    GCAAAATTGTTTTTGGTTGGATTGGAGAAGTAATCCTAGGGAAGGGTGGTGGAGCCAGTA2040    AATAGAGGAGTACAGTGTAAGCACCAAGCTCAAAGCGTGGACAGGTGTGCCGACAGAAGG2100    AACCAGCGTGTATATGAGGGTATCAAATAAAATTGCTACTACTTACCACC2150    __________________________________________________________________________

We claim:
 1. A method of inhibiting a CD4+ bearing malignant cell in amammal comprising administering to said mammal a therapeuticallyeffective amount of a lymphocyte chemoattractant factor (LCF) antagonistcomprising amino acids 1-15, 45-60, 65-85 or 115-130 of SEQ ID NO:1. 2.The method of claim 1, wherein said mammal is a human.
 3. The method ofclaim 1, wherein said CD4+ T cell is a lymphoma.
 4. The method claim 1,wherein said CD4+ T cell is a leukemia.
 5. The method of claim 1,further comprising administering to said mammal a chemotherapeutic agentin an effective dose which is lower than the standard dose when saidchemotherapeutic agent is used alone.
 6. A method for suppressing alymphocyte chemoattractant factor (LCF) CD4 interaction-mediatedphysiological response in a mammal comprising administering to saidmammal a fragment of LCF of SEQ ID NO:1 wherein said fragment inhibitsan LCF-CD4 interaction.
 7. The method of claim 6 wherein said fragmentreduces inflammation in said mammal.
 8. The method of claim 6 whereinsaid fragment reduces an immune response.
 9. The method of claim 8wherein said immune response is a cutaneous or respiratory late-phasereaction to an allergen, or is asthma.